Insulin resistance plays a major role in dyslipidemia, cardiovascular disease, and type 2 diabetes. TRB3, a mammalian tribbles homolog, whose chromosomal region 20p13-p12 has been linked to human type 2 diabetes, impairs insulin signaling through the inhibition of Akt phosphorylation and is overexpressed in murine models of insulin resistance. We here report that the prevalent TRB3 missense Q84R polymorphism is significantly (P < 0.05) associated with several insulin resistance-related abnormalities in two independent cohorts (n = 178 and n = 605) of nondiabetic individuals and with the presence of a cluster of insulin resistance-related cardiovascular risk factors in 716 type 2 diabetic patients (OR 3.1 [95% CI 1.2-8.2], P = 0.02). In 100 additional type 2 diabetic patients who suffered from myocardial ischemia, age at myocardial ischemia was progressively and significantly (P = 0.03) reduced from Q84Q to Q84R to R84R individuals. To test the functional role of TRB3 variants, either Q84 or R84 TRB3 full-length cDNAs were transfected in human HepG2 hepatoma cell lines. As compared with control HepG2 cells, insulin-induced Ser473-Akt phosphorylation was reduced by 22% in Q84- (P < 0.05 vs. control cells) and by 45% in R84-transfected cells (P < 0.05 vs. Q84 transfected and P < 0.01 vs. control cells). These data provide the first evidence that TRB3 gene plays a role in human insulin resistance and related clinical outcomes.