Prion protein (PrP C ) is a normal cellular glycoprotein that is expressed in almost all tissues including the central nervous system. Much attention has been focused on this protein because conversion of the normal PrP C to the diseased form (PrP Sc ) plays an essential role in transmissible spongiform encephalopathies such as mad cow disease and Creutzfeldt-Jakob disease. In spite of the extensive effort, the normal physiological function of PrP C remains elusive. Emerging evidence suggests that PrP C plays a protective role against cellular stresses including apoptosis induced by various pro-apoptotic agents such as Bax and staurosporine (STS), however, other reports showed overexpression of PrP C enhances STS-mediated apoptosis. In this study, we took a different approach by depleting endogenous PrP C using specific interfering RNA technique and compared the depleting and overproducing effects of PrP C on STS-induced apoptosis in neuro-2a (N2a) cells. We demonstrate here that down-regulation of PrP C sensitizes N2a cells to STS-induced cytotoxicity and apoptosis. The enhanced apoptosis induced by STS was shown by increased DNA fragmentation, immunoreactivity of Bax, and caspase-3 cleavage. We also showed that overproduction of PrP C had little or no effect on STS-mediated DNA fragmentation in N2a cells but it augments STS-mediated apoptosis in HEK293 cells, suggesting a cell line-specific effect. In addition, the inhibitory effect of PrP C on STS-mediated cellular stress appears to be modulated in part through induction of cell cycle G2 accumulation. Together, our data suggest that physiological level of endogenous PrP C plays a protective role against STS-mediated cellular stress. Loss of this protection could render cells more prone to cellular insults such as STS.