Background: The aim of this study was to investigate the effects of Atractylenolide-I (AT-I), a naturally occurring sesquiterpene lactone isolated from Atractylodes macrocephala Koidz, on human ovarian cancer cells. Material/Methods: The viability and anchorage-independent growth of ovarian cancer cells were evaluated using MTT and colony formation assay, respectively. Cell cycle and apoptosis were detected with flow cytometry analysis. The level of cyclin B1 and CDK1 was measured using qPCR and ELISA analysis. The expression of Bax, cleaved caspase-9, cleaved caspase-3, cytochrome c, AIF, and Bcl-2, and phosphorylation level of PI3K, AKT, and mTOR were determined with Western blot analysis. Results: AT-I decreased the cell viability and suppressed anchorage-independent growth of A2780 cells. Cell cycle was arrested in G2/M phase transition by AT-I treatment, which was related to decreased expression of cyclin B1 and CDK1 in a dose-dependent manner. In addition, the treatment induced apoptosis, as shown by up-regulation of Bax, cleaved caspase-9, cleaved caspase-3, and cytosolic release of cytochrome c and AIF, and downregulation of Bcl-2, in a dose-dependent manner. Then, the effects of AT-I on PI3K/Akt/mTOR pathways were examined to further investigate the underlying anti-cancer mechanism of AT-I, and the results showed that treatment with AT-I significantly decreased the phosphorylation level of PI3K, Akt, and mTOR. Conclusions: This study demonstrated that AT-I induced cell cycle arrest and apoptosis through inhibition of PI3K/Akt/mTOR pathway in ovarian cancer cells. These results suggest that AT-I might be a potential therapeutic agent in the treatment of ovarian cancer.