She made her acting debut in the Hindi film Doli Saja Ke Rakhna, directed by Priyadarshan, but it did not do well commercially. In a 2000 interview with India Today, Priyadarshan said that he “lapsed into depression” after the film flopped.

In 2003, she starred in Dhool with Vikram, Kaakha Kaakha with Suriya, Thirumalai with Vijay, all of which did well at the box office, Kaakha Kaakha, in particular, being one of the biggest hits of her career. She was listed amongst the Best Actress nominees in Filmfare Awards South for her roles in Dhool and Kaakha Kaakha. She got International Tamil Film Awards Special Jury For Best Actress. Dhool, Kaakha Kaakha and Thirumalai were amongst the top 10 films of the year and The Hindu wrote that she was “virtually the undisputed queen of Kollywood”. Actor Vikram referred to her as Lady Kamal Haasan due to her dedication in acting. In 2004, she acted in a double role in the critically acclaimed Perazhagan, opposite Suriya. She won Tamil Nadu State Best Actress Award for the film. In 2005, she acted in Manmadhan, opposite Silambarasan. She also performed the lead female role in the Telugu film Tagore, opposite Chiranjeevi, which was screened at the International Indian Film Academy Awards, and was a commercial success.

Jyothika married actor Suriya on 11 September 2006, with whom she was paired in seven films; Poovellam Kettuppar, Uyirile Kalanthathu, Kaakha Kaakha, Perazhagan, Maayavi, June R and Sillunu Oru Kaadhal. The couple has two children, daughter Diya (born 10 August 2007) and son Dev (born 7 June 2010).

Her most successful film to date is the Rajinikanth-starrer Chandramukhi 2005, in which she played the title role. She won accolades for her performance in Chandramukhi including a Tamil Nadu State Film Awards and Filmfare Awards Nomination. She was also offered a chance to act with Mohanlal in Udayon, directed by Bhadran, but she could not do it due to busy schedules in Tamil. Her first release in 2006 was Saravana, opposite Silambarasan after Manmadhan. Her role in June R was noticed before the release of the film. Jyothika’s performance is fine tuned and emotionally engaged. She has underplayed her emotions beautifully. Her films in 2006 include Vettaiyadu Villaiyadu, a crime-thriller film written and directed by Gautham Menon and Sillunu Oru Kaadhal, opposite Suriya.

Her last films were Gautham Menon’s Pachaikili Muthucharam and the critically acclaimed Mozhi, both in 2007. Her roles in these two films, as a negative character in the former (which she played for the first time) and as a deaf and mute in the latter earned her widespread appreciation. Among these, Mozhi won her acclaim. Her performance took her very close to winning the National Film Award for Best Actress. However, she lost the award by a narrow margin at the end. In Pachaikili Muthucharam she had a crucial role and pulled off a memorable performance. Her Malayalam films Raakilipattu and Seetha Kalyanam were released in 2007 and 2009 respectively after languishing in development hell for years. Jyothika later made a comeback through television adverts. In 2008, she appeared in a short film Herova? Zerova?. The film was written and produced by her father-in-law Sivakumar and also starred Vijay, R. Madhavan and Suriya.

Later in February 2016, director Bramma approached Jyothika to play the leading role in a “female-centric” script he had written and impressed by the offer, her husband Suriya agreed to finance the film. Jyothika subsequently participated in a series of workshops to get ready for her role in the film for twenty days. The movie is titled Magalir Mattum, written and directed by Bramma, in his second venture after Kuttram Kadithal (2015). It also features Saranya, Urvashi, Bhanupriya, Nassar and Livingston playing pivotal roles. Produced by Suriya, the venture began production in July 2016. She was also signed by Sri Thenandal Films for another project titled Mersal together in September 2016, opposite to Vijay, directed by Atlee A few days after the shoot began, Jyothika opted out of the project and the makers signed Nithya Menen to replace her. No official announcement is made so far about the replacement. In late February, Suriya confirmed Jyothika’s new project, Naachiyaar by sharing the first look posters of the film. She got several accloades for her performance in Naachiyar as Rough and Riveting Cop. Deccan chronicle addressed her as “Lady Singham”. As per behindwoods Jyothika is fantastic, as she masterfully plays the role of a menacing, short-tempered and a bold cop, with a gracious heart. Following the immense success of the film and Jyothika’s big fan following in the Telugu speaking states The film Naachiyar has been dubbed into Telugu by the title ‘Jhansi’. Jyothika agreed to portray a leading female role in Mani Ratnam’s project titled Chekka Chivantha Vaanam in early September 2017, and confirmed her participation to the media while promoting Magalir Mattum (2017). She had earlier appeared in Mani Ratnam’s production venture Dumm Dumm Dumm (2001), but stated her excitement at being selected in a film to be directed by him.

In 2018, she appeared as the lead role of Chitra, a housewife to Arvind Swamy’s character in Mani Ratnam’s film Chekka Chivantha Vaanam. The first look was released on 21 August. The film has a worldwide release on 27 September 2018.

Jyothika along with her husband Suriya’s Agaram Foundation donated ₹25 lakh to the Thanjavur government hospital on August 2020. Apart from the financial support she also donated medical equipment and beds. This comes after she was criticised for her comments on the poor maintenance of the hospital before.