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Kani joined The Serpent Players (a group of actors whose first performance was in the former snake pit of the zoo, hence the name) in Port Elizabeth in 1965 and helped to create many plays that went unpublished but were performed to a resounding reception.
These were followed by the more famous Sizwe Banzi is Dead and The Island, co-written with Athol Fugard and Winston Ntshona, in the early 1970s. He also received an Olivier Award nomination for his role in My Children My Africa!
Kani's work has been widely performed around the world, including New York, where he and Winston Ntshona won a Tony Award in 1975 for Sizwe Banzi Is Dead and The Island. These two plays were presented in repertory at the Edison Theatre for a total of 52 performances.
In 1987 Kani played Othello in a performance of Shakespeare's play in South Africa which was still under Apartheid. "At least I'll be able to kiss Desdemona without leaving a smudge." he said then. 
Nothing but the Truth (2002) was his debut as sole playwright and was first performed in the Market Theatre in Johannesburg. This play takes place in post-apartheid South Africa and does not concern the conflicts between whites and blacks, but the rift between blacks who stayed in South Africa to fight apartheid, and those who left only to return when the hated regime folded. It won the 2003 Fleur du Cap Awards for best actor and best new South African play. In the same year he was also awarded a special Obie award for his extraordinary contribution to theatre in the USA.
Kani is executive trustee of the Market Theatre Foundation, founder and director of the Market Theatre Laboratory and chairman of the National Arts Council of SA.
On 20 February 2010, Kani received Life Time award (SAFTA Awards). Kani has also received the Avanti Hall of Fame Award from the South African film, television and advertising industries, an M-Net Plum award and a Clio award in New York. Other awards include the Hiroshima Peace Culture Foundation Award for the year 2000 and the Olive Schreiner Prize for 2005. He was voted 51st in the Top 100 Great South Africans in 2004. In 2006, he was awarded an honorary doctorate by the University of Cape Town. Nelson Mandela Metropolitan University awarded him an honorary Doctor of Philosophy in 2013.
- Sizwe Banzi is Dead (1972) (co-authored with Athol Fugard and Winston Ntshona)
- The Island (1973) (co-authored with Athol Fugard and Winston Ntshona)
- Statements After an Arrest Under the Immorality Act (co-authored with Athol Fugard and Winston Ntshona)
- My Children My Africa! (actor)
- Nothing But the Truth (2002) (sole playwright)
- The Tempest (2008) (actor in the role of Caliban, at the Baxter Theatre, Cape Town; Courtyard Theatre, Stratford-upon-Avon; and tour of Richmond, Leeds, Bath, Nottingham, Sheffield)
Film and television
- The Wild Geese (1978)
- Marigolds in August (1980)
- Killing Heat (1981)
- The Grass Is Singing (1981)
- "Master Harold"...and the Boys (1985) (TV)
- Saturday Night at the Palace (1987)
- An African Dream (1987)
- Options (1988)
- A Dry White Season (1989)
- Othello (1989) (TV)
- The Native Who Caused All the Trouble (1989)
- An African Dream (1990)
- Sarafina! (1992)
- In Darkest Hollywood: Cinema and Apartheid (1993) (Non-fiction)
- Soweto Green (1995)
- The Ghost and the Darkness (1996)
- Kap der Rache (1997) (TV) (German)
- Kini and Adams (1997)
- The Tichborne Claimant (1998)
- Final Solution (2001)
- Hillside (2005–2008) (TV series)
- Silent Witness (Finding Rachel) (2008)
- Endgame (2009)
- White Lion (2010)
- Coriolanus (2011)
- Nothing But the Truth (2002)
- "Honorary doctorates for June graduation". Electronic Monday Paper (University of Cape Town). 2006-06-12. Retrieved 2006-08-07.[dead link] Vol 25 No 14
- "NMMU to honour John Kani". Port Elizabeth Herald. 2013-04-10. Retrieved 2013-04-17.