Heme oxygenase-1 (HO-1), a known cytoprotective enzyme implicated also in the cell cycle regulation and angiogenesis, exerts many of its beneficial effects through carbon monoxide (CO). We studied the roles of HO-1 and CO in cardiac regeneration after myocardial infarction. Prior to coronary artery ligation, male Wistar rats were given either cobolt protoporphyrin IX to induce HO-1 or CO-donor methylene chloride. Cardiac regeneration was assessed by immunohistochemistry and confocal microscopy. CO significantly increased the accumulation of c-kit+ stem/progenitor cells into the infarct area and induced formation of new coronary arteries by promoting a substantial differentiation of c-kit+ cells into vascular smooth muscle cells (c-kit+/GATA6+ cells). Furthermore, CO increased proliferation of cardiomyocytes in the infarct border area at 4weeks post-infarction. This suggests proliferation of newly formed cardiomyocytes derived from c-kit+ cells as 10% of c-kit+ cells expressed early cardiac marker Nkx2.5. Increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha), stromal cell derived factor-1alpha (SDF-1alpha) and vascular endothelial growth factor-B (VEGF-B) were found in the infarct areas of CO-donor pretreated hearts suggesting that these factors potentially promoted the migration of c-kit+ cells into the infarct area and subsequent vasculogenesis and myocardial regeneration by CO. HO-1 increased both capillary and vascular densities, while only a small increase of c-kit+ cells was found. HO-1 upregulated SDF-1alpha, but did not have effect on HIF-1alpha and VEGF-B. In conclusion, HO-1 and CO have differential roles and mechanisms of action in cardiac regeneration. Modulation of the HO-1/CO axis may provide a novel tool for the repair of cardiac injury.