Oxidative stress has been speculated to play an essential role in diabetic cardiomyopathy. This study was designed to examine the effect of the antioxidant catalase on diabetes-induced cardiomyocyte dysfunction and the cellular mechanisms involved. Adult wild-type (FVB) and transgenic mice with cardiac-specific overexpression of catalase were made diabetic by a single injection of streptozotocin (STZ, 220 mg/kg; i.p., maintained for two weeks). Cardiomyocyte contractile properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dL/dt), intracellular Ca(2+) level and decay rate. STZ depressed -dL/dt, prolonged TPS and TR(90), elevated resting intracellular Ca(2+) level and reduced intracellular Ca(2+) decay in FVB myocytes. While catalase exhibited little effect on contractile and intracellular Ca(2+) properties in control myocytes, it negated diabetes-induced cardiomyocyte mechanical abnormalities. Diabetic myocytes exhibited enhanced levels of reactive oxygen species and apoptosis, which were alleviated by catalase. Western blot analysis revealed that diabetes reduced Akt phosphorylation, enhanced the silent information regulator 2 (Sirt2), and upregulated Forkhead transcriptional factor Foxo3a as well as glycogen synthase kinase-3beta (GSK-3beta) and pGSK-3beta. While catalase itself exhibited little effect on these proteins or their phosphorylation (with the exception of Sirt2), it significantly attenuated diabetes-induced alteration in pAkt, Foxo3a and Sirt2 without affecting GSK-3beta. Inhibition of Sirt2 using splitomicin impaired cardiomyocyte contractile function (reduced PS, +/-dL/dt, prolonged TPS and TR(90)). In summary, our data suggest potential roles of Akt, Foxo3a and Sirt2 in the onset of diabetic cardiomyopathy and the therapeutic potential of catalase.