Fatty acid translocase cluster determinant 36 (CD36), which is part of the FFA transporter system, has been identified in several tissues such as muscle, liver, and insulin-producing cells. Several studies have reported that induction of CD36 increases uptake of FFA in INS-1 cells and Caco-2/15 cells, suggesting the functional interplay between glucose and FFA in terms of insulin secretion and oxidative metabolism. The lipotoxic effect of FFA on normal pancreatic beta-cells is induced only by the combined exposure to hyperglycemic conditions . It is also reported that hyperglycemic conditions induce the expression of CD36 in intestinal epithelial cells . However, we do not currently know the regulating mechanism and physiological role of CD36 on glucotoxicity in pancreatic beta-cells. Also, diverse mechanisms are involved in the pathogenesis of β-cell failure in type 2 diabetes. Of them, the accumulation of ceramide, a bioactive lipid metabolite, is suggested to play a major role in inflammatory and stress responses that induce diabetes. However, the downstream inflammatory target of ceramide has not been defined. Using rat islets and the INS-1 β-cell line, we hypothesized that activation of the redox sensitive protein TXNIP is involved in ceramide induced beta cell dysfunction. Incubation of INS-1 cells and primary islets with C2-ceramide (N-acetyl-sphingosine) down regulated insulin and PDX-1 expression and increased β-cell apoptosis. Ceramide treatment induced a time dependent increase in TXNIP gene expression accompanied by activation of nuclear factor (NF)-kB and reduced mitochondrial thioredoxin (TRX) activity. Pretreatment with sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of the scavenger receptor CD36, blocked ceramide-induced up regulation of TXNIP expression and activity of NF-kB. Blockade of NF-kB nuclear translocation by the peptide SN50 prevented ceramide mediated TXNIP induction. Furthermore, SSO also attenuated ceramide induced early loss of insulin signaling and apoptosis. Collectively, our results unveil a novel role of CD36 in early molecular events leading to NF-kB activation and TXNIP expression. These data suggest that CD36 dependent NF-kB-TXNIP signaling contributes to the pathogenesis of pancreatic β-cell dysfunction and failure.