It has been suggested that oxidative stress involving reactive oxygen species (ROS) induces granulosa cell apoptosis, leading to follicular atresia, and that T-lymphokine-activated killer cell-originated protein kinase (TOPK) suppresses cancer cell apoptosis induced by several stimuli. However, it remains to be determined whether TOPK affects oxidative stress-induced granulosa cell apoptosis. The present study demonstrates that TOPK inhibition increases human granulosa cOV434 cell apoptosis induced by hydrogen peroxide (H 2 O 2 ). co-treatment with the TOPK inhibitor, OTS514, in combination with H 2 O 2 increased p53 acetylation and its expression, whereas it decreased Sirtuin 1 (SIRT1) expression, contributing to the promotion of apoptosis. In addition, the SIRT1 activator, resveratrol, or the SIRT1 inhibitor, Ex527, reduced or elevated H 2 O 2 -induced cOV434 cell apoptosis, respectively. Furthermore, the p53 inhibitor, Pifithrin-μ, diminished the augmentation in poly(AdP-ribose) polymerase (PARP) cleavage induced by OTS514 plus H 2 O 2 , while the Mdm2 antagonist, Nutlin 3, increased PARP cleavage. Moreover, OTS514 further decreased the SIRT1 transcriptional activity decreased by H 2 O 2 , but promoted the H 2 O 2 -induced p53 or p21 transcriptional activity. Notably, the expression of exogenous p53 reduced SIRT1 transcriptional activity. Taken together, the findings of the present study demonstrate that TOPK inhibition promotes p53-mediated granulosa cell apoptosis through SIRT1 downregulation in response to H 2 O 2 . Therefore, it can be concluded that TOPK suppresses H 2 O 2 -induced apoptosis through the modulation of the p53/SIRT1 axis, suggesting a potential role of TOPK in the regulation of human granulosa cell apoptosis, leading to the promotion of abnormal follicular development.