The aim of this study was to elucidate cellular mechanisms involved in ceramide-induced apoptosis and its attenuation by hepatocyte growth factor (HGF). Human retinal pigmented epithelial cells (RPE) incubated with C2 ceramide accumulated reactive oxygen species (ROS) in mitochondria and underwent apoptosis in a dose-dependent manner. Ceramide-treated cells showed increased caspase-3 activation and an increase in mitochondrial membrane permeability transition (MPT). Low doses of H2O2 (100 μM) alone induced negligible apoptosis; however, ceramide-induced apoptosis was significantly enhanced by co-incubation with H2O2 (100 μM). Furthermore, ceramide treatment significantly decreased catalase enzymatic activity and protein expression. HGF pretreatment (20 ng/ml) significantly inhibited ceramide-induced apoptosis and reduced the accumulation of ROS, the activation of caspase-3, and the increase in MPT and prevented the reduction in catalase activity and expression. Together, the data suggest that ceramide induces apoptosis in RPE cells by increasing ROS production, MPT, and caspase-3 activation. The ceramide effect is potentiated by H2O2 and associated with a reduction in catalase activity, suggesting that catalase plays a central role in regulating this apoptotic response. The ability of HGF to attenuate these effects demonstrates its effectiveness as an antioxidant growth factor.