We found that TRIB3, [corrected] an endogenous inhibitor of Akt (PKB), is expressed in pancreatic beta-cells. The TRIB3 [corrected] expression is significantly increased in islets isolated from hyperglycemic Goto-Kakizaki rats compared with normal glycemic controls. In vitro high glucose treatment also resulted in increased TRIB3 [corrected] expression in rat INS1 cells. To investigate the role of TRIB3 [corrected] in the regulation of beta-cell function, we established an INS1 stable cell line allowing inducible expression of TRIB3. [corrected] We demonstrated that overexpression of TRIB3 [corrected] mimicked the glucotoxic effects on insulin secretion and cell growth in INS1 cells. Moreover, induction of TRIB3 [corrected] also synergistically enhanced high-glucose-elicited apoptosis in INS1 cells, whereas siRNA knock-down of TRIB3 [corrected] showed the opposite effects. We also confirmed that the DeltaPsim of mitochondria was decreased, caspase-3 activity was up-regulated and reactive oxygen species content was increased in TRIB3 [corrected] overexpressing beta cells in high glucose condition. Most interestingly, the oestrogen receptor (ER) stress inducer, thapsigargin, mimicked the high glucose effects on up-regulation of TRIB3 [corrected] and generation of apoptosis in cultured INS1 cells. These effects were specifically prevented by siRNA knock down of TRIB3. [corrected] We therefore conclude that TRIB3 [corrected] is implicated in glucotoxicity- and ER stress-induced beta-cell failure.TRIB3 [corrected] could be a potential pharmacological target for prevention and treatment of type 2 diabetes.