We found that TRIB3, in endogenous inhibitor of Akt (PKB), is expressed in pancreatic beta-cells. The TRIB3 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 expression in rat INS1 cells. To investigate the role of TRIB3 in the regulation of beta-cell function, we established an INS1 stable cell line allowing inducible expression of TRIB3. We demonstrated that overexpression of TRIB3 mimicked the glucotoxic effects Oil insulin secretion and cell growth in INS1 cells. Moreover, induction of TRIB3 also synergistically enhanced high-glucose-elicited apoptosis in INS1 cells, whereas siRNA knock-down of TRIB3 showed the opposite effects. We also confirmed that the Delta Psi m of mitochondria was decreased, caspase-3 activity was up-regulated and reactive oxygen species content was increased in TRIB3 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 and generation of apoptosis in cultured INS1 cells. These effects were specifically prevented by siRNA knock down of TRIB3. We therefore conclude that TRIB3 is implicated in glucotoxicity- and ER stress-induced beta-cell failure. TRIB3 could be a potential pharmacological target For prevention and treatment of type 2 diabetes.