This study aims to investigate whether miR-25 acts on regulating the expression of PTEN and affecting the proliferation and apoptosis of ovarian cancer cell line SKOV3. The expression of miR-25 and PTEN in the test group and the control group were compared. The targeting relationship between miR-25 and PTEN was identified by the experiment of dual-luciferase reporter gene. SKOV3 cells were cultured in vitro and were divided into five groups. Flow cytometer was used to detect the cell apoptosis and cycles and measure the expressions of miR-25 and PTEN. Western blot was used to measure the expression of proteins. The expression of miR-25 was increased and the expression of PTEN was decreased in the ovarian tumour tissues, compared to normal ovarian tissues. The expression of PTEN could be targeting inhibited by miR-25. The expression of p-AKT could be significantly inhibited by the down-regulation on the expression of miR-25 and (or) up-regulation on the expression of PTEN, and the phosphorylation of FoxO3a was significantly decreased, and the expression of p27Kip1 was increased. Moreover, cell apoptosis was significantly increased, and cell cycle was blocked at the stage G0/G1. Through targeting inhibition of PTEN, the abnormally over-expressed miR-25 can up-regulate the activity of PI3K/AKT signal pathway, increase the phosphorylation activity of FoxO3a and down-regulate the expression of p27Kip1, and consequently to antagonize the cell apoptosis of ovarian cancer cell SKOV3 and promote the progress of cell cycles. These may play roles in the onset of ovarian cancer.