Abstract: Objective To investigate the effects and mechanism of up-regulation of heme oxygenase-1 (HO-1) on cardiac function of diabetic rats. Methods Forty-eight wistar rats were divided into 3 groups: control group (group A, n=16), diabetic group (group B, n=16) and diabetic rats treated with HO-1 inducer cobalt-protoporphyrinⅨ(CoPP) to enhance HO-1 expression group (group C, n=16). Tail vein blood samples of glucose, insulin and adiponectin were measured in each group. Isolated hearts were prepared to measure the cardiac and coronary functions. Levels of malondialdehyde (MDA), malonyl coenzyme A (malonyl-CoA), acetyl coenzyme A (acetyl-COA) and superoxide anion (O^2-) in heart tissue were measured. The signaling molecule of cardiac tissue and HO activity level were measured by Western blot. Results The level of glucose in group B was significantly higher than in group A (P＜ 0.01), while the level of insulin (P＜ 0.01) and adiponectin (P＜ 0.05) in group B were lower than in group A. Isolated hearts from diabetic rats in Langendorff configuration displayed worse ventricular function and higher coronary resistance (CR) compared with hearts from control hearts, all the indexes had dramatically increased with the up-regulation of HO-1 on diabetic animals (P＜ 0.01), while the level of CR had decreased (P＜ 0.01). HO-1 inducer CoPP enhanced HO-1 protein levels and reduced oxidative stress in diabetic animals, which indicated by the decrease in heart O^2- and MDA levels (P＜ 0.05). HO-1 up-regulation increased signaling molecule level and reversed the eNOS/iNOS expression imbalance which was observed in the untreated diabetic heart (P＜ 0.05). Moreover, after the improvement of HO-1 expression, a rise in malonyl-COA as well as a decrease in acetyl-COA was observed in diabetic hearts (P＜ 0.05). Conclusion Up-regulation of heme oxygenase-1 can improve both cardiac function and coronary flow by blunting oxidative stress, restoring eNOS/iNOS expression balance via pAMPK-p-eNOS pathway, thereby favoring improvement in both endothelial function and insulin sensitivity.