Clinical studies have shown that nuclear expression of the inhibitor of apoptosis protein Survivin in tumor cells predicted a favorable prognosis whereas cytosolic-localized protein caused a decreased overall survival. Therefore Survivin's subcellular localization may be important for its anti-apoptotic capacity. To address this question, we investigated localization and function of Survivin in normal human lung fibroblasts (NHLFs) and HeLa tumor cells. NHLFs of early passages expressed Survivin in the nucleus and were highly sensitive to C2 ceramide, which induces the mitochondrial apoptotic pathway. In contrast, NHLFs at higher passages relocated Survivin to the cytosol and became more resistant to C2 ceramide. Blocking nuclear export of Survivin by leptomycin B in HeLa cells increased susceptibility to C2 ceramide. In addition, transduction of HeLa cells with Survivin fused to a nuclear localization signal augmented basal expression levels of p53 and Bax and enhanced sensitivity for intrinsic apoptosis. Those findings suggest that a predominant nuclear localization of Survivin increases the sensitivity for pro-apoptotic stimuli, whereas nuclear export enables Survivin to fulfill its inhibitor of apoptosis function. A therapeutic intervention which holds Survivin in the nucleus of tumor cells might improve cancer therapy.