Abstract: Objective To evaluate the effect of Foxo3a gene over-expression on the development of rat ovarian granulosa cells and in prevention of cisplatin-induced ovarian damage in rats. Methods Rat ovarian granulose cells released mechanically from the ovaries were cultured in vitro and identified with HE staining and immunohistochemical staining for FSHR. A recombinant adenovirus carrying Foxo3a gene was constructed for infecting the granulose cells, and the cell growth and expressions of cyclin D1, p27, Bax, and Bim were detected; the cell apoptosis and cell cycle changes were detected using Hoechst/PI 33342 staining and flow cytometry, respectively. The transfected cells were challenged with cisplatin and the cell apoptosis was detected with flow cytometry. Results Over 90% of the cultured cells survived and contained more than 95% ovarian granulose cells. Infection of the cells with the recombinant adenovirus resulted in over-expressions of Foxo3a at the mRNA and protein levels at 36 h and 48 h after the infection, respectively. The infected cells showed suppressed proliferation, increased apoptotic rate and cell cycle arrest in G1 phase with increased expressions of Bim, p27, and cyclin D1 but without significant changes in Bax expression. Cisplatin exposure caused a significantly higher apoptosis rate in the infected cells than in the control cells. Conclusion Over-expression of Foxo3a gene can promote granulose cell apoptosis by increasing Bim expression and cause cell cycle arrest in G1 phase by increasing cyclin D1 and p27 expressions, but can not prevent the toxic effects of cisplatin on ovarian granulosa cells.