Objective: Cluster determinant 36 (CD36), a fatty acid transporter, was reported to have a pivotal role in glucotoxicityinduced β-cell dysfunction. However, little is known about how glucotoxicity influences CD36 expression. So, the predominant objective is to reveal the role of RAC1-NOX in CD36 activation and its impact for the β-cell dysfunction during diabetes mellitus. Methods: Insulinoma cell line INS-1 cells and primary rat islets were treated to high glucose (30mM) for 24 hours in the absence or presence of RAC1 inhibitor NSC23766. RAC1 and NOX activity was assessed using the RAC1 Activation Assay kit (Merck Millipore) and by chemiluminescence assay. Reactive oxygen species and mitochondrial membrane potential were measured by using DCFDA and DIOC6 dye respectively. Moreover, the protein expression level of CD36, MAPK signaling pathways in response to high glucose is assessed by western blot analysis Results: Exposure of INS-1 cells to high glucose upregulated RAC1 and NADPH oxidase activation, and induced apoptosis. Interestingly, upregulated RAC1 and NADPH oxidase induced CD36 expression. These effects of high glucose were significantly decreased in INS-1 cells treated with NSC23766. Moreover, RAC1 inhibition by NSC23766 significantly reduced high glucose-induced mitochondrial dysfunction assessed by measuring the mitochondrial membrane potential and cytochrome c release. Inhibition of RAC1 also attenuated MAPK signaling. Conclusion: RAC1 regulates NOX activity, thereby increasing the expression of CD36 and its downstream signaling lead to β-cell dysfunction under high glucose conditions.