Alteration of glycosphingolipid (GSL) expression plays key roles in the pathogenesis and pathophysiology of many important human diseases, including cancer, diabetes and glycosphingolipidosis. Inflammatory processes are involved in development and progression of diabetic nephropathy, a major complication of type 2 diabetes mellitus. GSLs are known to play roles in inflammatory responses in various diseases, and levels of renal GSLs are elevated in mouse models of diabetic nephropathy; however, little is known regarding the pathophysiological role of these GSLs in this disease process. We studied proinflammatory activity of GSLs in diabetic nephropathy using spontaneously diabetic mouse strain KK. Mice were fed a high-fat diet (HFD) (60% kcal from fat) or normal diet (ND) (4.6% kcal from fat) for a period of 8 wk. HFD-feeding resulted in quantitative and qualitative changes of renal globo-series GSLs (particularly Gb3Cer), upregulation of TNF-α, and induction of renal inflammation. Gb3Cer/Gb4Cer treatment enhanced inflammatory responses via TLR4 in TLR4/MD-2 complex expressing cells, including HEK293T, mouse bone marrow-derived macrophages (BMDMs) and human monocytes. Our findings suggest that HFD-induced increase of Gb3Cer/Gb4Cer positively modulate TLR4-mediated inflammatory response, and that such GSLs play an important pathophysiological role in diabetic nephropathy.