Ceramide is known to play a role in cell signaling pathway involved in apoptosis. Most studies suggest that enhanced ceramide generation is the result of hydrolysis of sphingomyelin by sphingomyelinases. However, the role of ceramide synthase in enhanced ceramide generation has not been previously examined in hypoxia-reoxygenation injury. In the present study, we demonstrated that 60 min hypoxia of rat renal tubular epithelial NRK-52E cells in a gas chamber with 95% N₂ - 5% CO₂ with glucose deprivation resulted in a significant increase in ceramide generation. The ceramide level further increased after reoxygenation for 60 min. Exposure of cells to hypoxia-reoxygenation resulted in a significant increase in ceramide synthase activity without any significant change in sphingomyelin content as well as activity of acid or neutral sphingomyelinase. The hypoxia-reoxygenation of NRK-52E cells was also associated with the release of endonuclease G (EndoG) from mitochondria to cytoplasm measured by Western blotting and endonuclease activity assay. It further led to the fragmentation of DNA, and cell death. Specific inhibitor of ceramide synthase, fumonisin B1 (50 µM), suppressed hypoxia-reoxygenation-induced ceramide generation, and provided protection against hypoxia-reoxygenation-induced EndoG release, DNA fragmentation and cell death. Taken together, our data suggest that hypoxia-reoxygenation results in an activation of ceramide synthase rather than sphingomyelinase, and that ceramide synthase-dependent ceramide generation is a key modulator of EndoG-mediated cytotoxicity in hypoxia-reoxygenation injury to renal tubular epithelial cells.