Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolarix kaempferi Gordon (Pinaceae). Recent studies have reported that PAB exhibits cytotoxic effects in several cancer cell lines. In the present study, we assessed its antitumor activity and molecular mechanisms in HO-8910 and A2780 ovarian cancer cells in vitro. We found that PAB reduced cell viability and induced apoptosis in a dose- and time-dependent manner in HO-8910 and A2780 human ovarian cancer cells. The induction of apoptosis was also accompanied by the regulation of Bcl-2 and XIAP family proteins, cytochrome c and Apaf-1. Moreover, we observed that PAB treatment resulted in the activation of caspase-3 and -9, which may partly explain the anticancer activity of PAB. Collectively, the present study for the first time suggests that PAB enhances apoptosis of HO-8910 and A2780 cells through regulation of Bcl-2 and IAP family proteins. Moreover, the triggering of caspase-3 and -9 activation mediated apoptotic induction. Our results suggest that PAB may be a new therapeutic option for the treatment of ovarian cancers.