- Sharlto Copley as Wikus van de Merwe, a mild-mannered, shy, bumbling, awkward bureaucrat at the MNU Department of Alien Affairs, who becomes infected with an alien fluid, slowly turning him into one of the "prawns". This was the first time acting professionally in a feature film for Copley, a friend of director Blomkamp.[9]
- Jason Cope as Christopher Johnson, a District 9 prawn who assists Wikus in fighting MNU.
- Cope also performed the role of Grey Bradnam, the UKNR Chief Correspondent and all the speaking aliens, as well as for the cameraman Trent[10]
- David James as Colonel Koobus Venter, an aggressive, sadistic, and xenophobic PMC mercenary-soldier sent to capture Wikus. He is shown as taking pleasure in killing the aliens and responding brutally to anyone who opposes him.
- Vanessa Haywood as Tania Smit-van de Merwe, Wikus's wife.
- Mandla Gaduka as Fundiswa Mhlanga, Wikus's assistant and trainee during the eviction
- Eugene Wanangwa Khumbanyiwa as Obesandjo, a paralyzed psychopathic Nigerian gang leader who believes that eating alien body parts will enable him to operate their weapons
- Louis Minnaar as Piet Smit, managing director of MNU South Africa and Wikus's father-in-law
- Kenneth Nkosi as Thomas, an MNU security guard and good friend of Wikus
- William Allen Young as Dirk Michaels, the CEO of MNU
- Nathalie Boltt as Sarah Livingstone, a sociologist at Kempton Park University
- Sylvaine Strike as Katrina McKenzie, a doctor from the Department of Social Assistance
- John Sumner as Les Feldman, a MIL engineer
- Nick Blake as Francois Moraneu, a member of the CIV Engineer Team
- Jed Brophy as James Hope, an officer with the SAPS Alien Crimes Unit
- Vittorio Leonardi as Michael Bloemstein, MNU Department of Alien Civil Affairs
- Johan van Schoor as Nicolaas van de Merwe, Wikus's father
- Marian Hooman as Sandra van de Merwe, Wikus's mother
- Jonathan Taylor as the Doctor
- Stella Steenkamp as Phyllis Sinderson, MNU Alien Relations
- Tim Gordon as Clive Henderson, an entomologist at WLG University
- Nick Boraine as Lieutenant Weldon, Colonel Venter's right-hand man
- Robert Hobbs as Ross Pienaar, an MNU mercenary
- Trevor Coppola as MNU Mercenary
- Morne Erasmus as MNU Medic
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