Prohibitin (Phb1) is a highly conserved mitochondrial protein that is associated with granulosa cell differentiation, atresia, and luteolysis. Although prohibitin has been implicated in the suppression of apoptosis in mammalian cells, its specific role in programed cell death (PCD) in granulosa cells is unknown. In the present study we examined the role of prohibitin in mediating staurosporine and serum withdrawal induced apoptosis in undifferentiated rat granulosa cells. Treatment of granulosa cells isolated from immature rat ovaries with staurosporine and/or serum withdrawal induced a rapid decrease in the transmembrane potential of mitochondria, resulting in increased prohibitin content and induced apoptosis in a time- and dose-dependent manner. Infection of granulosa cells with a Phb1 adenoviral construct resulted in overexpression of prohibitin that markedly attenuated the ability of staurosporine and serum withdrawal to induce apoptosis via the intrinsic apoptotic pathway. To determine the site of action of Phb1, granulosa cells were transfected with a prohibitin-eGFP fusion construct and the fusion protein expression patterns were analyzed by fluorescence microscopy and Western blot analysis of cell fractionated samples. These studies indicated that the prohibitin-eGFP fusion protein moved from the cytoplasm into the mitochondria. However, no prohibitin-eGFP fusion protein was observed in the nucleus in response to the staurosporine-induced apoptotic stimulus. This result was corroborated by Western blot analysis with GFP-specific antibody. Furthermore, the prohibitin-eGFP fusion protein also inhibited PCD. These results provide evidence that prohibitin could serve an anti-apoptotic role in undifferentiated granulosa cells.