Transient reduction in coronary perfusion pressure in the isolated mouse heart increases microvascular resistance (paradoxical vasoconstriction) by an endothelium-mediated mechanism. To assess the presence and extent of paradoxical vasoconstriction in hearts from normal and diabetic rats and to determine whether increased heme oxygenase (HO)-1 expression and HO activity, using cobalt protoporphyrin (CoPP), attenuates coronary microvascular response. Male Wistar rats were rendered diabetic with nicotinamide/streptozotocin (NA/STZ) for 2 weeks and either CoPP or vehicle was administered, by intraperitoneal injection, daily for 3 weeks (0.5 mg/100 gm body wt). The isolated beating non-working heart was submitted to transient low perfusion pressure (20 mm Hg) and coronary resistance (CR) was measured. During low perfusion pressure, CR increased and was associated with increased lactate release. In diabetic rats, CR was higher, HO-1 expression and endothelial nitric oxide synthase (eNOS) were downregulated, and inducible nitric oxide synthase (iNOS) and superoxide anion (O2-) were upregulated. After 3 weeks of CoPP treatment, HO activity was significantly increased in the heart. Upregulation of HO-1 expression and HO activity by CoPP resulted in the abolition of paradoxical vasoconstriction and a reduction in oxidative ischemic damage. In addition, there was a marked increase in serum adiponectin. Elevated HO-1 expression was associated with increased expression of cardiac eNOS, B-cell leukemia/lymphoma extra long (Bcl-xL) and phospho activator protein kinase (pAKT) levels, and decreased levels of iNOS and malondialdehyde (MDA). These results suggest a critical role for HO-1 in microvascular tone control and myocardial protection during ischemia in both normal and mildly diabetic rats through the modulation of constitutive and inducible nitric oxide synthase expression and activity, and an increase in serum adiponectin.