Sphingolipids are membrane lipids and play critical roles in signal transduction. Ceramides, central components of sphingolipid metabolism, are involved in cell death. However, the mechanism of ceramides regulating cell death in plants remains unclear. Here, we found ceramides accumulated in mitochondria of accelerated cell death 5 mutant (acd5), and expression of mitochondrion-localized ceramide kinase (ACD5) suppressed mitochondrial ceramides accumulation and the acd5 cell death phenotype. We applied immuno-electron microscopy and observed ceramide-hyperaccumulation in acer acd5 double mutants which are characterized by mutations in both ACER (alkaline ceramidase) and ACD5 genes. The results confirmed that, plants with specific ceramide accumulation exhibited localization of ceramides to mitochondria, resulting in an increase of mitochondrial reactive oxygen species. Interestingly, when compared to the wild type, autophagy deficient mutants showed stronger resistance to ceramide-induced cell death. Lipid profiling analysis demonstrated that plants with ceramide accumulation exhibited a significant increase in phosphatidylethanolamine levels. Further, we found that ceramide treatment or endogenous ceramide accumulation induces autophagy. When exposed to exogenous ceramides, we found an increase in the level of ATG8e associated with mitochondria, where it bound to ceramides directly. Taken together, we proposed that the accumulation of ceramides in mitochondria can induce cell death by regulating autophagy.