Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). We hypothesized that inducible NOS (iNOS) plays a crucial role in LV remodeling after MI depending on its coupling status. MI was created in wild-type (WT), iNOS-knockout (iNOS(-/-)), endothelial NOS-knockout (eNOS(-/-)), and neuronal NOS-knockout (nNOS(-/-)) mice. iNOS and nNOS expressions were increased after MI associated with an increase in nitrotyrosine (NT) formation. The area of myocardial fibrosis and LV end-diastolic volume and ejection fraction were more deteriorated in eNOS(-/-) mice compared to other genotypes of mice 4 weeks after MI. The expression of GTP cyclohydrolase was reduced, and tetrahydrobiopterin (BH4) was depleted in the heart after MI. Oral administration of sepiapterin after MI increased dihydrobiopterin (BH2), BH4 and BH4/BH2 ratio in the infarcted but not sham-operated heart. The increase in BH4/BH2 ratio was associated with inhibition of NT formation and an increase in nitrite plus nitrate (NOx). However, this inhibition of NOS uncoupling was blunted in iNOS(-/-) mice. L-NAME abrogated sepiapterin-induced increase in NOx and angiogenesis, and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH4 synthesis and increasing bioavailable NO predominantly derived from iNOS. Sepiapterin increased capillary density and prevented LV remodeling and dysfunction after MI in WT, eNOS(-/-) and nNOS(-/-) but not iNOS(-/-) mice. L-NAME abrogated sepiapterin-induced increase in NOx and angiogenesis, and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH4 synthesis and increasing bioavailable NO predominantly derived from iNOS.