Mos Def began his music career in 1994 with the short-lived group Urban Thermo Dynamics with his younger brother DCQ, and his younger sister Ces. Despite their contract with Payday Records, the group only released two singles and the group's debut album, Manifest Destiny, did not see the light of day until 2004 when it was released through Illson Media. In 1996, he emerged as a solo artist and worked with De La Soul and da Bush Babees, before he released his own first single, "Universal Magnetic", which was a huge underground hit.

After signing with Rawkus Records, Mos Def and Talib Kweli formed the group, Black Star, and released a full length album under the name, Mos Def and Talib Kweli are Black Star. The album was released in 1998 and featured Hi-Tek producing most of the tracks.

Mos Def released his solo debut also on Rawkus Records, Black on Both Sides, in 1999 Def was also featured on Rawkus' influential Lyricist Lounge and Soundbombing series compilations. After the collapse of Rawkus, he and Kweli signed on to Interscope/Geffen Records, which released his second solo album, The New Danger, in 2004. Mos Def has drawn some criticism from his fan base about "keeping it real," after he appeared in a commercial that endorsed the GMC Denali sport utility vehicle. Mos Def released True Magic, his last solo album on Geffen Records, on December 29, 2006.

Grammy Award nominations were announced for 2007, and "Undeniable" was nominated for Best Rap Solo Performance . "Ghetto Rock" from his 2004 album The New Danger, was also nominated at the 2005 Grammy's

Mos began his professional acting career at the age of 14, appearing in the TV movie God Bless the Child, starring Mare Winningham. He then played the oldest child in the short-lived family sitcom, You Take the Kids, starring Nell Carter and Roger E. Mosley. His most notable acting role before his music career was that of Bill Cosby's sidekick on the short-lived detective show, The Cosby Mysteries.

After brief appearances in Bamboozled and Monster's Ball, Mos re-invigorated his acting career with his performance as a talented rapper who is reluctant to sign to a major label in Brown Sugar. He was nominated for an Image Award and a Teen Choice Award.

Notably in 2002, he played the role of Booth in Suzan-Lori Parks' Topdog/Underdog, a Tony-nominated and Pulitzer-winning Broadway play. He also received positive notices as the quirky Left Ear in the blockbuster hit, The Italian Job.

He has also appeared on Comedy Central's Chappelle's Show.

In 2004, he hosted the MOBO awards in London.

He has hosted the award-winning HBO spoken word show, Def Poetry since its inception. The show's fifth season aired in 2005.

Mos won Best Actor, Independent Movie at the 2005 Black Reel Awards for his portrayal of the skeptical Detective Sgt. Lucas in The Woodsman. For his portrayal of Vivien Thomas in HBO's film, Something the Lord Made, he was nominated for an Emmy Award, a Golden Globe, and won the Image Award. He then landed the role of Ford Prefect in the 2005 movie adaptation of The Hitchhiker's Guide to the Galaxy.

In 2006, Mos Def appeared in Dave Chappelle's Block Party alongside fellow Black Star companion Talib Kweli, while also contributing to the film's soundtrack. Also, Mos Def was featured as the black banjo player in the infamous "Pixie Sketch" from Chappelle's Show: The Lost Episodes. He was later edited out of it on the dvd. Additionally, Mos Def is in the motion picture 16 Blocks along with co-stars Bruce Willis and David Morse. During the first season of Boondocks, Mos was given the opportunity of starring as "Ganstalicious". He is also set to be in Toussaint, a film about Haitian revolutionary Toussaint L'Ouverture, opposite Don Cheadle and Wesley Snipes.He made a cameo appearance in the movie Talladega Nights: The Ballad of Ricky Bobby.

By the early 1990s, a brand of socially conscious hip hop that had been popularized by A Tribe Called Quest, KRS-One, and many others had been eclipsed in popularity by gangsta rap. Mos Def, as well as Talib Kweli, Common, Little Brother, Quannum Projects and others helped socially aware rap music experience something of a comeback in the late 1990s and early 2000s. Mos Def's collaboration with Talib Kweli, Mos Def and Talib Kweli are Black Star, was released during the aftermath of the deaths of 2Pac and The Notorious B.I.G. and focused on violence and deceit in hip-hop, in collaboration with other acts that did the same. Def's music also has references to his Islamic faith, and his contention that black artists receive little credit for their role in the birth of rock and roll music.

On Mos Def's 2004 album The New Danger, the rapper took his penchant for experimentation to a new level. Most of the songs were more hip-hop flavored stylings of blues and rock, with fewer raps thrown in. This threw off fans who were expecting another full-blown rap album. The New Danger also featured the controversial song, "The Rape Over", a parody of Jay-Z's The Blueprint hit "The Takeover":

Old white men is runnin' this rap shit,
Corporate forces runnin' this rap shit,
Some tall Israeli is runnin' this rap shit,
We poke out our asses for a chance to cash in.
Cocaine, is runnin' this rap shit,
'Dro, 'yac and e-pills is runnin this rap shit,
MTV is runnin' this rap shit,
Viacom is runnin' this rap shit,
AOL and Time Warner runnin' this rap shit,
Quasi - homosexuals is runnin' this rap shit.
High-placed executives temporarily made Mos take the song off releases of the album, citing clearance issues with Jay-Z and The Doors, a band which the song samples. The song has been called anti-Semitic for its veiled reference to Jewish record executive Lyor Cohen (the "tall Israeli" who then was head of the The Island Def Jam Music Group), and homophobic for its reference to "quasi-homosexuals."

Mos and Immortal Technique released a similarly controversial song, "Bin Laden" in 2005, which blamed the Reagan Doctrine and President George W. Bush for the September 11, 2001 attacks. A club remix song, featuring Eminem, was released the following year, in 2005.

In September 2005, Mos Def released the single "Katrina Clap", renamed "Dollar Day (Surprise, Surprise)" for the upcoming True Magic, (utilizing the instrumental for New Orleans rapper Juvenile's "Nolia Clap"), a criticism of the Bush administration's response to the Hurricane Katrina disaster. On the night of the 2006 MTV VMA's, Mos Def performed the single in front of a crowd on the streets. However, he was soon arrested for performing without a permit.