The 68-year-old Jayalalithaa along with 28 others including her trusted lieutenant O Pannerselvam took the oath of office and secrecy in the name of God in Tamil that was administered by Governor K Rosaiah.
The AIADMK supremo retained 15 of her ministers who were in the previous cabinet and 13 new faces including three women.
DMK treasurer M K Stalin, former DMK Ministers EV Velu, Ponmudy, party MLAs Sekhar Babu, Vagai Chandrasekhar and Ku Ka Selvam also attended the ceremony. For decades both AIADMK and DMK leaders had kept away from the oath-taking ceremonies of rivals.
Amid slogans of "Puratchi Thalaivi Amma Vazhga (Long live revolutionary leader Amma) by AIADMK supporters, Jayalalithaa stepped into the decorated Madras University Auditorium in her trademark green saree.
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Slogans hailing the party chief also echoed the venue when she signed the documents after taking oath.
While Jayalalithaa took oath alone, others were sworn-in in groups in a brief ceremony lasting less than 30 minutes.
At the venue, she was welcomed by top state government officials led by Chief Secretary K Gnanadesikan with a bouquet of flowers.
Later, when Governor Rosaiah arrived she welcomed him with a bouquet and introduced her Cabinet colleagues in keeping with the protocol.
Ministers in the outgoing Cabinet Natham Viswanathan, Vaithilingam, newly elected MLAs, Lok Sabha and Rajya Sabha MPs of the ruling party were also amongst the audience.