While studying at college, Nayanthara worked part-time as a model. She was spotted by Director Sathyan Anthikkad, who had seen some of her modelling assignments and approached her to play a pivotal role in his film Manassinakkare (2003). Although she turned down the offer initially, as she was not interested in films, she gave in eventually and agreed to do "just that one film". Manassinakkare went on to become a high financial success and she continued to receive acting offers. Both her releases in 2004, Natturajavu by Shaji Kailas, and Fazil's psychological thriller Vismayathumbathu, saw her co-starring alongside Mohanlal; while she played the protagonist's adopted sister in the former, she portrayed a ghost in the latter. Her performance in Vismayathumbathu, in particular, was lauded, with critics claiming that she had "stolen the thunder with her author-backed role", and was "the revelation of the film". She also acted in Thaskara Veeran and Rappakal.

Nayanthara subsequently started appearing in Tamil and Telugu films. In 2005, she was cast in Hari's Ayya, debuting in the Tamil film industry. Behindwoods.com stated that she had made a "sensational debut in Tamil", while a reviewer from Nowrunning.com said that her "presence with her beautiful smile is crowd winning". While still shooting for Ayya, she was selected for the comedy horror film Chandramukhi, after its Director P. Vasu's wife had seen Manassinakkare and recommended her. The film ran for over 800 days in theatres, eventually turning Nayanthara into one of the most-sought after actresses in Tamil. Later that year she appeared in A R Murugadoss' Ghajini, in which she played a secondary female character. Nayanthara then accepted to appear in an item number as herself in the Perarasu-directed masala film Sivakasi.

Nayanthara was in a relationship with actor Simbu, with whom she worked in Vallavan (2006). In 2010, Prabhu Deva wife Latha filed a petition at the family court, seeking directions against Deva from living in a relationship with Nayanthara and requesting a reunion with him. Furthermore, she threatened to go on hunger strike if Prabhu Deva married Nayanthara, while several women's organisations conducted protests against Nayanthara for bringing disrepute to the Tamil culture, burning an effigy of her. In 2012, Nayanthara confirmed that she had ended her relationship with Deva. She is currently in a relationship with Director Vignesh Shivan—with whom she worked in Naanum Rowdy Thaan—which became official when they walked in together at an awards show in Singapore.

Nayanthara regained her star billing in Kollywood acting in the Vishnuvardhan-directed gangster film Billa (2007). A remake of the same-titled 1980 Tamil film, it went on to become a success at the box office, while Nayanthara received many accolades for her performance as Sasha in her new glamour look. Sify heaped praise on her, describing her as a "show stopper". The reviewer further wrote: "Nayanthara has gone full throttle to look her sexiest best [...] She has a beautiful body which she flaunts daringly [...] and is also able to bring out the cold aloofness and bitterness of her character. Similarly a critic from Nowrunning.com noted that she looked "great in mini-skirts, jacket, dark glasses and tall boots".

In 2008 she had five releases, four of which were in Tamil. Her first release was the family entertainer Yaaradi Nee Mohini. Behindwoods.com wrote: "Nayan dispels her bombshell act and proves that she can more than just that. She breaks into tears when needed, shows vicious contempt when rubbed the wrong way in the name of love, and looks endearing in songs". Nowrunning.com stated that she "exhibits her acting skills in full measure [and] gives a moving performance in emotional scenes", while Sify's critic wrote that she looked "sensational and has done a great job in perhaps the meatiest role she has done so far". She won the Vijay Award for Favourite Heroine for the second consecutive year, and was nominated for the Best Actress prize at the 56th Filmfare Awards South.

Her subsequent releases were Kuselan, Satyam, Villu and Aegan . In 2009 she released Aadhavan.

For the Malayalam movie Elektra (2010) Nayanthara dubbed her lines for the first time. Then she dubbed for the Telugu film Krishnam Vande Jagadgurum, Tamil films Iru Mugan,Naanum Rowdy Dhaan and Dora.

Nayanthara was born and brought up as a Syrian Christian. On 7 August 2011, she converted to Hinduism at the Arya Samaj Temple in Chennai. She has gone through Shuddhi Karma, a procedure involving a Vedic purification and a Homam where Nayanthara chanted the hymns from Veda and Gayatri Mantra under the guidance of the priest. After the purification ceremony, a Certificate of Conversion to Hinduism was issued to her and her stage name, Nayanthara, became her official name. She also visited a number of holy places, including Haridwar and Rishikesh in May 2014.

Choosing to make a comeback, Nayanthara began shooting for Krish's Krishnam Vande Jagadgurum (2012) after an eleven-month sabbatical. She portrayed the role of a Journalist and her performance was praised, with a reviewer from Sify stating that "Nayanthara does not play the normal commercial glam-doll for a change and she is good" and "her chemistry with Rana is perfect". The following year, she won critical acclaim for her performance in Atlee's romantic drama Raja Rani (2013), winning both the Filmfare and Vijay Award for Best Actress. Playing the role of a wife in an unhappy marriage, a critic from Sify.com noted, "Nayanthara gets a well-written role and she couldn`t ask for a better character than Regina for her return after a three-year hiatus... she looks good, throws herself into the role, approaches her part with restraint and minimalist fuss, and turns in a sincere performance." Sify.com also said her role was "unquestionably the film's biggest strength". Likewise, another reviewer from Behindwoods.com stated the role was "Nayanthara’s career best performance till date" and it was a "great comeback by the Actress with a role which gave her ample scope".

Her next release, Vishnuvardhan's Arrambam (2013), featuring Ajith Kumar in the lead, also won acclaim with a reviewer from Rediff stating that "Nayanthara looks gorgeous and does full justice to her character", while Behindwoods's critic stated that "Nayanthara puts out a fiery face when required and also showcase her oomph and style in many scenes, adding "this superstar continues to shine". Both films performed well commercially and were amongst the highest grossing Tamil films of 2013.

Nayanthara's rising popularity as an Actress led to her being cast in female-centric films, a genre considered a rarity in South Indian cinema. Sekhar Kammula cast her in the titular role of his bilingual Anaamika (2014), a remake of the Hindi film Kahaani (2012), in which Nayanthara portrayed an IT professional who moves to Hyderabad to look for her missing husband. Sify.com labelled her performance in the film "a one-woman show by her in a difficult role... she nails the character perfectly whether it in the initial scenes showing her anguish or in the stunning climax, where she takes a brave decision". In contrast, Rediff.com stated that her "performance is lacklustre in the first half".

Along with strong character roles, Nayanthara continued to appear in action and comedy films, including the successful Bhaskar the Rascal (2015) and Thani Oruvan (2015), during the same period. She then went on to win her second Filmfare Award for her role of a deaf and mute girl seeking revenge in Vignesh Shivan's black comedy Naanum Rowdy Dhaan (2015). About her performance, Sify.com's reviewer noted "Nayanthara's Kadambarai is a well-etched out role, the Actress is effortlessly charming and she does full justice", and "infuses the part with the right portions of vulnerability, restraint, and quiet strength, delivering a performance that is nothing short of perfect". Following the success of the film, Nayanthara was referred to in the media as the "Best Actresses".

In her first release of 2016, Nayanthara portrayed a rape victim in Puthiya Niyamam (2016) opposite Mammooty, with a critic from the IBTimes stating "Nayantara's role as Vasuki Iyer is one of her career's best until now and her decision to dub in her own voice for her character is worthy of appreciation". She continued to play diverse roles, appearing as a village-based school Teacher in Thirunaal (2016), a spy in the commercially successful Vikram-starrer Iru Mugan (2016) and the queen of a fantasy kingdom in Kaashmora (2016). In 2017, she will appear in a series of female-centric films, where she plays the main lead. Her first release is likely to be the horror film Dora, which will be followed by the social drama Aramm, where she portrays a district collector. She also plays the main character in Chakri Toleti's Kolaiyuthir Kaalam, the thriller Imaikkaa Nodigal and Bharath Krishnamachari's Ner Vazhi, amongst other upcoming releases.