Refractory wound is a severe complication that leads to limb amputation in diabetes. Endothelial nitric oxide synthase (eNOS) plays a key role in normal wound repair but is uncoupled in streptozotocin (STZ)-induced type 1 diabetes because of reduced cofactor tetrahydrobiopterin (BH₄). We tested the hypothesis that overexpression of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for de novo BH₄ synthesis, retards NOS uncoupling and accelerates wound healing in STZ mice. Blood glucose levels were significantly increased in both male endothelium-specific GTPCH I transgenic mice (Tg-GCH; via a tie-2 promoter) and wild-type (WT) littermates 5 days after STZ regimen. A full-thickness excisional wound was created on mouse dorsal skin by a 4-mm punch biopsy. Wound closure was delayed in STZ mice, which was rescued in STZ Tg-GCH mice. Cutaneous BH₄ level was significantly reduced in STZ mice vs. WT mice, which was maintained in STZ Tg-GCH mice. In STZ mice, constitutive NOS (cNOS) activity and nitrite levels were decreased compared with WT mice, paralleled by increased superoxide anion (O₂⁻) level and inducible NOS (iNOS) activity. In STZ Tg-GCH mice, nitrite level and cNOS activity were potentiated and O₂⁻ level and iNOS activity were suppressed compared with STZ mice. Thus endothelium-specific BH₄ overexpression accelerates wound healing in type 1 diabetic mice by enhancing cNOS activity and suppressing oxidative stress.