Cardiac insulin resistance is a key pathogenic factor for diabetic cardiomyopathy, but its mechanism remains largely unclear. Here we demonstrated that diabetes significantly inhibited cardiac Akt phosphorylation from 2 weeks to 2 months in wide-type (WT) mice, but not in cardiac-specific metallothionein-transgenic (MT-TG) mice. Cardiac Akt2 expression and phosphorylation was decreased and insulin-induced cardiac Akt2 and GSK-3β phosphorylation and glycogen synthase dephosphorylation were also decreased in WT, but not MT-TG, diabetic mice. Deletion of the Akt2 gene either in vitro H9c2 cells or in vivo significantly impaired cardiac glucose metabolic signaling. In addition, diabetes significantly increased cardiac Akt negative regulator tribbles (TRB)3 expression only in WT mice, suggesting the possible contribution of MT inhibition of diabetic up-regulation of TRB3 to Akt2 function preservation. Cardiac H9c2 cells with and without forced MT-overexpression (MT-H9c2) were treated with tert-butyl hydroperoxide (tBHP), which significantly reduced Akt2 phosphorylation in both basal and insulin-stimulating conditions only in H9c2 cells. Silencing TRB3 expression with SiRNA completely prevented tBHP’s inhibition of insulin-stimulated Akt2 phosphorylation in H9c2 cells, while overexpression of TRB3 in MT-H9c2 cells completely abolished MT preservation of insulin-stimulated Akt2 phosphorylation. Forced-overexpression of TRB3 by adenovirus-mediated gene delivery in MT-TG hearts also abolished MT’s preservation of cardiac insulin signaling and prevention of diabetic cardiomyopathy. These results suggest that diabetes-attenuated cardiac Akt2 function via up-regulating TRB3 plays a critical role in diabetic inhibition of insulin signaling in the heart. MT preserved cardiac Akt2-mediated insulin signaling by inhibiting TRB3, leading to the prevention of diabetic cardiomyopathy.