Microcystin-LR (MCLR) is one of the most common and most toxic members of the microcystins, which cause serious environmental disasters worldwide.Although the major toxicity of MCLR has been ascribed to its potent ability to inhibit protein phosphatase 1 and protein phosphatase 2A (PP2A), recent studies have suggested that MCLR may also perturb other important cellular processes, such as generation of ceramide.Ceramide is an essential second messenger in cells and regulates various cellular mechanisms, including PP2A activation and cytoskeleton destabilization.However, whether and how ceramide may mediate MCLR-induced cellular effects is unclear.We have previously reported that low concentrations of MCLR upregulate, rather than inhibit, PP2A activity in human embryonic kidney 293 (HEK293) cells.In this study, we provide evidence that MCLR induces ceramide generation in HEK293 cells and in mouse kidney.Furthermore, ceramide may mediate the MCLR-induced upregulation of PP2A activity and protein level of PP2A regulatory subunits in HEK293 cells.MCLR intoxication also causes the PP2A/B55α subunit to localize to the Golgi apparatus, and this process may also be mediated by ceramide.Importantly, ceramide may mediate cytoskeleton destabilization, cell detachment, and apoptosis induced by MCLR in HEK293 cells, whereas a ceramide synthase inhibitor, desipramine, protects cells from these changes.Our results suggest that ceramide may mediate MCLR-induced PP2A regulation and cytoskeleton destabilization.