Apoptosis of stem cells may be related to certain degenerative conditions such as progressive tissue damage and an inability to repair. Ceramide induces cell death in various cell types. However, the underlying mechanisms of ceramide-induced cell death in stem cells are not explored. This study was designed to investigate the cell death process caused by cell-permeable ceramide and to determine the underlying mechanisms in mesenchymal stem cells derived from human adipose tissue (hASCs). Ceramide caused a loss of cell viability in a concentration- and time-dependent manner, which was largely attributable to apoptosis. Ceramide induced generation of reactive oxygen species (ROS) and disruption of the mitochondrial membrane potential. The ROS generation caused by ceramide was prevented by the antioxidant N-acetylcysteine (NAC). Although ceramide induced release of cytochrome c from mitochondria and activation of caspase-3, the ceramide-induced cell death was partially prevented by caspase inhibitors. Addition of ceramide caused apoptosis-inducing factor (AIF) nuclear translocation, which was prevented by antioxidant. Taken together, these data suggest that ceramide induces cell death through both caspase-dependent and caspase-independent mechanisms mediated by ROS generation in hASCs.