| Character | Voice Actor | Notes |
| Agent J | Keith Diamond | Agent J (James Edwards) is one of the main agents of the organization. He is energetic, and tries to bring emotion back to the bland organization. At one point, J arrests the alien Jarra for attempting to steal the Earth's ozone layer and sell it on the Black Market. He remains somewhat of a sidekick to Agent K. He is referred as "Slick" by Agent K. Throughout the series, most of the other agents treat J like a total screw-up who never should have been allowed in the MIB. |
| Agent K | Ed O'Ross in Season 1, Gregg Berger in Seasons 2–4 | Agent K (Kevin Brown) is a top agent and a founder of the organization; and after working at the organisation for nearly 40 years, he shows nearly no emotion. He and J must constantly fight off his old partner Alpha. |
| Agent L | Jennifer Lien in Seasons 1–3, Jennifer Martin in Season 4 | Agent L (Laurel Weaver) is a morgue worker who joins the MIB after she helps Agent K and J stop Edgar the Bug from stealing The Galaxy. She stays in the medical section of the organization until Season 4, wherein she becomes a field agent with Agent X as her partner. |
| Chief Zed | Charles Napier | The head of the organization and K and J's boss. He is usually shown in his office, high above the main floor of the headquarters. It is known that Zed took the job of director at some point prior to the incident with Serlena and the Light of Zartha in 1978. |
| Frank the Pug | Eddie Barth | Frank the Pug is an extraterrestrial living in New York City, in the guise of a pug. It is later revealed that the "human" at Frank's newspaper stand is a robot, and that Frank's true form resembles a pug, albeit with a dark green color, antennas, and a three-pointed tail. |
| The Worms | Patrick Pinney and Pat Fraley | The Worms are four insect-like aliens who love to drink coffee and relax in the organization's break room, all of their names are Neeble, Geeble, Sleeble and Mannix. They are renowned for knowing many figures of speech and the organization rulebook. |
| Jack Jeebs | Tony Shalhoub in Season 1, Billy West in Seasons 2–4 | Jack Jeebs is a humanoid alien who runs a pawn shop, carrying both alien and human merchandise. Jeebs' head is frequently blasted off, but he possesses regenerative abilities that let him grow his head back within seconds, much to his annoyance. |
| Idikiukup and Bob | Patrick Pinney and Pat Fraley | A pair of alien twins (earning them the nickname of the Twins) that serve as the MIB's head scientists along with Agent L. They mostly stay at headquarters though Bob pilots a helicopter in "It's a Fmall Fmall World". |
| Dr. Zan'dozz Zeeltor | Steve Kehela | A blue-skinned humanoid alien who replaces Elle as chief scientific officer of the organization. He is very eccentric about the simple items around Earth. Most of his inventions usually cause more harm around J, such as a sticky note pad that started to constrict him. Zeeltor has a pet named Lucy: a Futavian bacteria colony. |
| Agent X | Adam Baldwin | A big-eared, green-skinned alien from the G-Dan galaxy; recruited into the organization as Elle's partner, to smooth over human-alien relations. Though he has an impeccable record, X was suspended six times in his home galaxy. |
| Alpha | David Warner | Alpha is Agent K's former mentor, and now the sworn enemy of him and the M.I.B. Stealing a Verulian Cosmic Integrator, he used it to graft stolen alien body parts onto himself until he was defeated and captured. Escaping and allying himself with Vangus and the Ixions, Alpha turned himself into a cyborg. Alpha is killed in the series finale, "The Endgame Syndrome". |
| Agent D | John Mariano | Agent D (Derrick Cunningham) is Agent K's former partner who is now retired after getting too old in the fight against the evil aliens. He was a founding member of the organization. Derrick is seen enjoying his retirement with his wife, before he is wiped out of existence by a time traveling alien-hating bigot, and restored by Agent K and a Cerebro-enhanced Agent J. |
| Edgar the Bug | N/A; originally portrayed by Vincent D'Onofrio in the film | A cockroach-like alien and the main antagonist of the feature film who was killed by Agent L. His only appearance in the series was in a flashback to his death in "The Big Bad Bug Syndrome". Edgar is revealed to have a twin brother named Edwin as well as being the Bug Queen's favorite. His death causes The Queen to put a bounty on L’s head. |
| Edwin the Bug | Vincent D'Onofrio | The twin brother of Edgar who was sent by the Bug Queen (along with his siblings Dung the dung beetle and Moe the mosquito) to capture Agent L for killing Edgar and deliver her to the queen in exchange for her royal jelly as a award, however he was defeated by the Worms who tricked his siblings to use De-Atomizers to kill him. |
| Geen | Vincent D'Onofrio | A voracious Bug who was imprisoned in M.I.B intergalactic prison. Geen and several other inmates were temporarily released by Doctor Lupo and proceeded to wreak havoc upon the MIB HQ until they were rounded up by K and J. |
| The Bug Queen | Mary Kay Bergman | The queen of Edgar and Edwin's planet and mother of all bugs that resembles a praying mantis. The Bug Queen puts a bounty on Agent L's head, offering her royal jelly to any Bug that brings her Elle. She later attempted to colonize Manhattan with a new generation of offspring but was foiled by the MIB. The Bug Queen subsequently attempted to conquer Earth once more but was foiled by Agent K and Agent J who flooded the tunnel which the Bugs were using as a base. She was ultimately vanquished by the combined efforts of K and a temporarily amnesiac J. |
| Arquillians | N/A | The Arquillians are a race of tiny humanoid aliens. Arquillians have large heads and eyes, but a small body, and hide in human-like robots. The Arquillians own the Arquillian Galaxy, a miniaturized source of sub-atomic power, and will not stand for it being lost or stolen. They are ruled by a complex monarchy. |
| Baltians | N/A | A tall, slender species that vaguely resemble Grey aliens and were the first species encountered by the original Men in Black. The Baltians return to Earth every ten years to provide new alien technologies to help fund the organisation. |
| Buzzard | Sherman Howard | A Zombarian bounty hunter who is an occasional adversary of Agents J and K. Howard and D'Onofrio both previously were guests on Miami Vice and were enemies of Crockett and Tubbs like they were Agent J and K. |
| Dak Jeebs | Billy West | Jack Jeebs' criminal big brother who often picks on him and calling him squirt, especially blowing up his head just to frustrate him. Unlike his brother, he actually enjoys the sensation when getting dismembered. |
| Drekk | Ron Perlman | Drekk is alien who has a long criminal record: "longer than Van Allen's belt", according to Chief Zed. He is a former cell-mate of Frank the Pug, and an antagonist of the lead characters. |
| The Emperor Worm | Vincent D'Onofrio | The Emperor Worm is the leader of the Worms. Unlike the other Worms, the Emperor is hugely fat, and almost double the height of the others. It is also revealed that only royalty are allowed to drink coffee on their home planet, resulting in the Worms to avoid drinking any of it under his presence. He usually shows up un-announced at the organisation. The Emperor is careless and defenseless, so the other Worms must watch him. |
| Agent H | Kurtwood Smith | One of the founding members of the Men in Black. He was responsible for designing most of the organization's medical equipment. |
| Agent Q | N/A | One of the founding members of the organization; responsible for the weapons and vehicles that the organization use. |
| Aileen | Beth Broderick | A female alien and agent of the Men in Black: a blue-skinned humanoid with extendable, tentacle-like fingers. She lost her former partner to an alien shapeshifter called an Inanimate. She is later assigned another partner named Eidi, whom she treats similarly to K's treatment of J. Like K, she is a legendary agent on her home planet. |
| Eidi | N/A | Aileen's rookie partner, who belongs to the same species as her and was assigned to work with her after the capture of the Inanimate. Eidi and J bond while working the case due to their shared status as novices partnered with the planet's best agents. |
| Agent U | N/A | An average agent. His name before joining the organization was Upton, which J easily deduces. There is a running gag where U's agent codename and the word "you" are often confused. Agent U is shown to be a major suck up and simply teaches alien children how to disguise themselves as humans. |
| Agent E | N/A | An agent who works in Hollywood, getting work for aliens in movies and television shows. |
| Troy The Symbiote | Rino Romano | A young adventurous symbiote who has a playful relationship with J. His dream is to work for the organization, much to his mother's distress. Troy becomes an MIB intern in "The Heads You Lose Syndrome". |
| Klah'Mikk | N/A | A paperwork filing alien who works for the organisation. |
| The Fmeks | N/A | The warmongering counterparts of the Arquillians. The Fmeks are the Arquillian's sworn enemy because the Arquillians' planet is bigger than their home world Fmoo. |
| The Ixions | N/A | A race of oil-loving creatures, who with the help of their leader, Vangus, attempt to plunder the Earth's oil to sell it on the intergalactic market. They were ultimately defeated by MIB, along with Alpha. |
| Vangus | Billy West | The leader of the Ixions. |
