Background - Estrogen is vasoprotective in animal models of vascular injury, yet the mechanisms involved are incompletely understood. The role of inducible nitric oxide synthase (iNOS) in vascular repair is controversial, but many lines of evidence indicate that it plays a role in neointima formation after arterial injury and that 17β-estradiol (E2) modulates iNOS expression. This study tested the hypothesis that E2 reduces neointima formation after vascular injury via a mechanism that is dependent on modulation of iNOS expression. Methods and Results - Male and female wild-type (iNOS+/+) mice and mice with homozygous deletion of the iNOS gene (iNOS-/-) were studied intact (INT) or after ovariectomy (OVX) and implantation of E2 or vehicle (V) pellets. Mice were randomized to 8 groups based on sex, iNOS status, OVX, and treatment with E2 or V. Twenty-eight days after carotid artery ligation, mice were euthanized, and occluded vessels were evaluated for neointima formation by morphometric analysis. There was a marked sexual dimorphism in neointima formation in both the iNOS+/+ mice and the iNOS-/- mice. iNOS+/+ INT females had a >90% reduction in neointima formation compared with iNOS+/+ males, and iNOS-/- INT females had a 65% reduction in neointima formation compared with iNOS-/- males. The sexually dimorphic response was attenuated by OVX and restored by E2 replacement in both iNOS+/+ and iNOS-/- mice. Conclusions - These results demonstrate that the vasoprotective effects of E2 after ligation vascular injury are, at least in part, independent of iNOS expression.