Objective: Recent studies have suggested that non-TG metabolites of free fatty acids cause insulin resistance and lipotoxic tissue injury. Sphingolipids, including ceramide and its derivatives, are recognized as important signal mediators in the regulation of inflammation, apoptosis, proliferation, and differentiation. Podocytes are characterized by a prominent nucleus, a well-developed Golgi system and endoplasmic reticulum, and large numbers of mitochondria. A recent study has reported that podocyte mitochondria play a primary role in maintaining energy homeostasis. However, the role of podocyte mitochondrial dysfunction in kidney pathology is incompletely understood. Thus we hypothesize that mitochondrial ceramide accumulation in podocytes induces cell damage and later albuminuria. Methods: Immortalized mouse podocytes were used as a model to evaluate the effects of ceramide on intracellular ROS generation and podocyte autophagy. OLETF rats were used as an animal model of type 2 DM. Ceramide and ROS were measured by LC-MS/MS, and fluorescence microscopy, respectively. Transmission electron microscopy was used for more detailed ultrastructural analysis of the mitochondrial morphology. Results: Cultured podocytes exposed to putative risk factors [high glucose, fatty acid, and angiotensin II in combination, GFA] showed an increase in ceramide accumulation and ROS generation in podocyte mitochondria. Pretreatment with myriocin reversed GFA-induced mitochondrial ROS generation and cell death. Myriocin-pretreated cells were protected from GFA-induced mitochondrial fragmentation. Finally, OLETF rats treated with myriocin (0.3 mg/kg/day) from 18 to 22 weeks of age showed a profound decrease in albuminuria. Furthermore, podocyte injury and the histologic changes characteristic of diabetic nephropathy were not observed after myriocin treatment. Conclusion: In this study, we showed that mitochondrial ceramide accumulation could result in damage of podocyte through ROS production and impaired autophagy in DKD mouse model and this signaling pathway could be used as a pharmacologically target to abate the development of DKD.