Kirron Kher made her film debut in 1983 in the Punjabi feature film Aasra Pyar Da and received rave reviews for her performance. She soon took a hiatus from films following her divorce to raise her son, Sikander Kher.
Meanwhile, she worked for her second husband Anupam Kher as a costume designer and appeared in one film in between, Pestonjee in 1988, wherein she acted alongside Anupam Kher.
Her return to acting came about with theatre, with the play Saalgirah, by playwright Javed Siddiqui and directed by Feroz Abbas Khan. Then she hosted three television shows, starting with Purushkshetra on Zee TV which got her much acclaimed for bringing out the discussion of sexuality to the forum for the first time, while highlighting women's issues at the same time, Kiron Kher Today and Jagte Raho with Kiron Kher, before doing Bollywood films.
Her comeback film was by Shyam Benegal, Sardari Begum (1996), which won her the Special Jury Award at the 1997 National Film Awards.
In 2000, she appeared in film director Rituparno Ghosh's Bengali film Bariwali 1999, to critical acclaim. When she won the National Film Award for Best Actress for the film, a controversy arose as a Bangla film actress, Rita Koiral, claimed that she had dubbed for the character of Kirron, making her an equal claimant to the award. Kirron refuted the charges claiming she spent hours rehearsing for her dialogue delivery, and the award was eventually not shared.
In 2002, Kirron appeared in Devdas (2002), alongside Shahrukh Khan, Madhuri Dixit and Aishwarya Rai, and was nominated for the Filmfare Best Supporting Actress Award.
In Khamosh Pani (Silent Waters) (2003), a film that portrays the plight of a woman abducted during partition of India, her character not only refused to kill herself as suggested by her family, but marries her abductor and, after his death, makes an earning teaching Quran to local children. It shows how her life changes dramatically when her son takes up Islamic extremism in 1979 during the rule of Zia-ul-Haq and his process of Islamization of Pakistan.She won the Best Actress Award, at Locarno International Film Festival, Switzerland, the Karachi International Film Festival, Karachi, and International Festivals at Ciepie in Argentina,and Cape Town in South Africa while the film won the Best Film – Golden Leopard, Festival Grand Prize at Locarno.
The Indian Film Festival of Los Angeles (IFFLA) 2004 paid tribute to Kirron during its annual festival.
In October 2004 Kirron made a guest appearance, along with her husband, in the American TV series ER playing Parminder Nagra's mother, Mrs. Rasgotra, in episode "Damaged."
In 2005, she essayed the role of role of Sunanda in the tele-serial Prratima on Sahara One channel, before which she had appeared in TV series like Dil Na Jaane Kyon (Zee TV), Isi Bahane, and Chausath Panne.
Despite playing mostly supporting roles, her successful movies include Main Hoon Na (2004), Hum Tum (2004), Veer-Zaara (2004), and Mangal Pandey: The Rising (2005), where her performance drew rave reviews. Her work in Rang De Basanti (2006) was a huge hit and her performance got her second nomination for the Filmfare Best Supporting Actress Award. Her role in Fanaa (2006) and Kabhi Alvida Naa Kehna (2006) also received appreciation. Adding to her list of comical roles in 2008 she appeared in Singh is Kinng, Saas bahu aur Sensex and Dostana.
In 2009, she appeared as judge on India's Got Talent, the Indian franchise of the Got Talent series.