He first rose to prominence for portraying the titular role in Henry: Portrait of a Serial Killer (1986), and is best known for starring as Merle Dixon in the AMC series The Walking Dead (2010–2013) and as Yondu Udonta in Guardians of the Galaxy (2014), followed by its sequels Guardians of the Galaxy Vol.

He is a recurring collaborator of Guardians of the Galaxy director and co-CEO of DC Studios James Gunn, appearing in all of his films to date including Slither (2006), Super (2010) and The Suicide Squad (2021).

Rooker's other notable roles include Chick Gandil in Eight Men Out (1988), Frank Bailey in Mississippi Burning (1988), Terry Cruger in Sea of Love (1989), Rowdy Burns in Days of Thunder (1990), Bill Broussard in JFK (1991), Hal Tucker in Cliffhanger (1993), Sherman McMaster in Tombstone (1993), Jared Svenning in Mallrats (1995), Detective Edward Kennesaw in Deceiver (1997), Detective Howard Cheney in The Bone Collector (1999), Detective Jake Riley in Replicant (2001), and Buddy in F9 (2021).

[10][11] In June 2010, Rooker revealed via Twitter that he was to appear in the AMC television series The Walking Dead as Merle Dixon, one of the survivors of a zombie apocalypse.

[12][13] Rooker is also known for his roles in video games such as Call of Duty: Black Ops (where he played himself), Mike Harper in Call of Duty: Black Ops 2 in November 2012, and as Merle in The Walking Dead: Survival Instinct, the video game based on the television series.