Objective: Sepiapterin reductase (SPR) catalyzes the final step of tetrahydrobiopterin (H₄B) biosynthesis, and the first step of H₄B regeneration from an exogenous precursor sepiapterin. Despite the potential significance of SPR in regulating H₄B-dependent nitric oxide (NO^•) production, the endothelium-specific sequence and functions of SPR remain elusive. Methods and results: We first cloned endothelial SPR cDNA from bovine aortic endothelial cells (Genebank: DQ978331). In cells transiently transfected with SPR, SPR activity (HPLC) was dramatically increased by 19-fold, corresponding to a significant increase in endothelial H4B content (HPLC) and NO· production (electron spin resonance). In vivo delivery of SPR gene significantly increased vascular SPR protein expression (mouse vs. bovine antibodies to differentiate endogenous vs. exogenous), activity, H₄B content and NO^• production, as well as NO^•-dependent vasorelaxation. In endothelial cells transfected with siRNA specific for SPR, approximately 87% of mRNA was attenuated (real time quantitative RT-PCR), corresponding to a significant reduction in SPR protein expression and activity, which was associated with decreases in both intracellular H4B content and NO^• level. Exogenous administration of sepiapterin to endothelial cells significantly upregulated H4B and NO^• levels, which were attenuated by SPR RNAi. H₄B-stimulated increase in NO^• production however, was SPR RNAi-independent. GTP cyclohydrolase 1 (GTPCH1) expression and activity, as well as dihydrofolate reductase (DHFR) expression, were not affected by SPR RNAi, whereas DHFR activity was significantly downregulated. Conclusions: These data represent the first to study endothelial SPR functionally, and clearly demonstrate an important role of endothelial SPR in modulating H₄B and NO^• bioavailability.