Objective: Beta cell dysfunction contributes to the development and progression of type 1 (T1D) and type 2 diabetes (T2D). In this study, we evaluated the role of Rac1, a small GTPase protein in streptozotocin (STZ) induced beta cell toxicity and the underlying mechanisms. Methods: We subjected INS-1 cells and human pancreatic 1.1b4 beta cells to STZ (1-2mM) for 8 & 24 hours respectively. Rac1 activation was measured by non-radioactive Rac1 activation kit (Millipore). NADPH oxidase activity was measured by Lucigenin based chemiluminescence assay. PKC-δ activation and nuclear translocation was measured by western blot & immunofluorescence analysis. Results: Exposure of INS-1 cells or human pancreatic 1.1b4 beta cells with STZ-stimulated the expression of active Rac1-GTP (3.5 fold p < 0.001) compared with control cells. STZ treatment elevated NADPH oxidase activity (3 fold p < 0.001) correlated with a loss of mitochondrial membrane potential ΔΦ followed by mitochondrial release of cytochrome c into the cytosol leads to caspase -3 activation. Further, caspase-3 activity mediates the proteolytic cleavage of PKC-δ catalytic fragment which then moved to the nucleus and potentiated the STZ - induced INS-1 and human pancreatic 1.1b4 beta cell apoptosis evaluated by TUNEL-assay. Interestingly, pharmacological inhibition of Rac1-GTP using NSC23766 (50uM) or small interference RNA (siRNA) blocked ST2-induced Rac1-GTP activation with a reduction of NADPH oxidase activity. This effect was associated with inhibition of mitochondrial dysfunction with a reduction in PKC-δ nuclear accumulation and cell apoptosis (p < 0.005). In addition, exposure of INS-1 cells or human pancreatic 1.1b4 beta cells to VAS2870 (20uM), an NADPH oxidase inhibitor blocked ST2-induced PKC-δ activation and INS-1 and human pancreatic 1.1b4 beta cell apoptosis. Conclusion: Rac1-GTP signaling contributes to STZ induced beta cell toxicity through NADPH oxidase dependent PKC-δ signaling. Thus, inhibition of Rac1-GTP signaling may promote beta cell survival under STZ-induced beta cell toxicity.