Chronic exposure to free fatty acids (FFAs) may induce beta cell apoptosis in type 2 diabetes. However, the precise mechanism by which FFAs trigger b cell apoptosis is still unclear. Tribbles homolog 3 (TRB3) is a pseudokinase inhibiting Akt, a key mediator of insulin signaling, and contributes to insulin resistance in insulin target tissues. This paper outlined the role of TRB3 in FFAs-induced INS-1 beta cell apoptosis. TRB3 was promptly induced in INS-1 cells after stimulation by FFAs, and this was accompanied by enhanced INS-1 cell apoptosis. The overexpression of TRB3 led to exacerbated apoptosis triggered by FFAs in INS-1-derived cell line and the subrenal capsular transplantation animal model. In contrast, cell apoptosis induced by FFAs was attenuated when TRB3 was knocked down. Moreover, we observed that activation and nuclear accumulation of protein kinase C (PKC) delta was enhanced by upregulation of TRB3. Preventing PKC delta nuclear translocation and PKC delta selective antagonist both significantly lessened the pro-apoptotic effect. These findings suggest that TRB3 was involved in lipoapoptosis of INS-1 beta cell, and thus could be an attractive pharmacological target in the prevention and treatment of T2DM.