A major target of cadmium (Cd²⁺) toxicity is the kidney proximal tubule (PT). Cd²⁺-induced apoptosis of PT cells is mediated by sequential activation of calpains at 3-6 hours and caspases-9 and -3 after 24 hours exposure. Calpains also partly contribute to caspase activation, which emphasizes the importance of calpains for PT apoptosis by Cd²⁺. Upstream processes underlying Cd²⁺-induced calpain activation remain unclear. We describe for the first time that 10-50 µM Cd²⁺ causes a significant increase in ceramide formation by ~22% (3 hours) and ~72% (24 hours), as measured by a diacylglycerol kinase assay. Inhibition of ceramide synthase with fumonisin B₁ (3 µM) prevents ceramide formation at 3 hours and abolishes calpain activation at 6 hours, which is associated with significant attenuation of apoptosis at 3-6 hours using Hoechst 33342 nuclear staining and/or MTT death assays. This indicates that Cd²⁺ enhances de novo ceramide synthesis and calpains are a downstream target of ceramides in apoptosis execution. Moreover, addition of C₆-ceramide to PT cells increases cytosolic Ca²⁺ and activates calpains. Apoptosis mediated by C₆-ceramide at 24 hours is significantly reduced by caspase-3 inhibition, which supports cross-talk between calpain- and caspase-dependent apoptotic pathways. We conclude that Cd²⁺-induced apoptosis of PT cells entails endogenous ceramide elevation and subsequent Ca²⁺-dependent calpain activation, which propagates kidney damage by Cd²⁺.