Objective : Altered metabolism has been implicated in the pathogenesis of beta-cell failure in type 2 diabetes. Of them, many clinical studies have confirmed a positive correlation between plasma and tissue levels of several ceramide species play a major role in inflammatory and stress responses that induce type 2 diabetes. Here we investigated the role of CD36 signaling and its impact for the beta-cell inflammation induced by ceramide. Methods : To address this question, we used INS-1, rat islets and human l.lb4 pancreatic beta-cells with C2-ceramide (N-acetyl-sphingosine). Gene expression was assessed by real-time RT-PCR. Rac1 activation was measured by non-radioactive Rac1 activation kit (Millipore). NADPH oxidase activity was measured by Lucigenin based chemiluminescence assay. Protein expression levels of Src, Vav2, TXNIP and insulin signaling were measured by western blot. Results : Exposure of INS-1 and human l.lb4 beta cells to C2-ceramide (50 uM) induced a time dependent increase in Src mediated Racl-GTP activation by Vav2 tyrosine phosphorylation. Racl-GTP activation enhanced NADPH oxidase activity leads to nuclear factor NF-kB mediated upregulation of TXNIP mRNA. Interestingly, pharmacological inhibition of CD36 by Sulfo-N-succinimidyl oleate (SSO) blocked Src activated Vav2 GEF activity. Further, reduced expression of active Racl-GTP resulted in decreased NADPH oxidase activity. Under these same conditions, nuclear factor NF-kB transcriptional activity was strongly inhibited. Moreover, inhibition of CD36 downregulate TXNIP mRNA 〈 0.05) and upregulate the Insulin, PDX1 mRNA levels < 0.05). Finally, inhibition of CD36 by SSO reduced INS-1 and human Pancreatic l.lb4 beta cell apoptosis. Conclusion : Ceramide triggers the CD36 mediated upregulation of Vav2 guanine nucleotide exchange factor (GEF) activity by Src activation. This is associated with enhanced Rac1-GTP- NADPH oxidase activity with cell apoptosis. Collectively, our results unveil a novel role of CD36 in early molecular events leading to the ceramide-induced pathogenesis of pancreatic beta-cell dysfunction and failure.