The signaling of glycogen synthase kinase-3beta (GSK-3beta) has been implicated in stress-induced apoptosis. However, the pro-apoptotic role of GSK-3beta is still unclear. Here, we show the involvement of GSK-3beta in ceramide-induced mitochondrial apoptosis. Ceramide induced GSK-3beta activation via protein dephosphorylation at serine 9. We previously reported that ceramide induced caspase-2 and caspase-8 activation, Bid cleavage, mitochondrial damage, and apoptosis. In this study, we found that caspase-2 activation and the subsequent apoptotic events were abolished by the GSK-3beta inhibitors lithium chloride and SB216763, and by GSK-3beta knockdown using short interfering RNA. We also found that ceramide-activated protein phosphatase 2A (PP2A) indirectly caused GSK-3beta activation, and that the PP2A-regulated PI 3-kinase-Akt pathway was involved in GSK-3beta activation. These results indicate a role for GSK-3beta in ceramide-induced apoptosis, in which GSK-3beta acts downstream of PP2A and the PI 3-kinase-Akt pathway, and upstream of caspase-2 and caspase-8.