Apoptosis is meticulously controlled in living organisms. Its dysregulation has been shown to play a key role in a number of human diseases, including neoplastic, cardiovascular, and degenerative disorders. Bcl-2 family member proteins and inhibitors of apoptosis proteins are two major negative regulators of apoptosis. We report here the characterization of novel antiapoptotic protein, fortilin, which we identified through yeast two-hybrid library screening. Sequence analysis of fortilin revealed it to be a 172-amino acid polypeptide highly conserved from mammals to plants. Fortilin is structurally unrelated to either Bcl-2 family member proteins or inhibitors of apoptosis proteins. Northern blot analysis showed the fortilin message to be ubiquitous in normal tissue but especially abundant in the liver, kidney, and small intestine. Western blot analysis using anti-fortilin antibody showed more extensive expression in cancerous cell lines (H1299, MCF-7, and A549) than in cell lines derived from normal tissue (HEK293). Immunocytochemistry using HeLa cells transiently expressing FLAG-tagged fortilin and immunohistochemistry using human breast ductal carcinoma tissue and anti-fortilin antibody both showed that fortilin is predominantly localized in the nucleus. Functionally, the transient overexpression of fortilin in HeLa cells prevented them, in a dose-dependent fashion, from undergoing etoposide-induced apoptosis. Consistently, U2OS cells stably expressing fortilin protected the cells from cell death induced by etoposide over various concentrations and durations of exposure. In addition, fortilin overexpression inhibited caspase-3-like activity as assessed by the cleavage of fluorogenic substrate benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin. Furthermore, the antisense depletion of fortilin from breast cancer cell line MCF-7 was associated with massive cell death. These data suggest that fortilin represents a novel antiapoptotic protein involved in cell survival and apoptosis regulation.