The two known estrogen receptors, ERα and ERβ, mediate the effects of estrogen in all target tissues, including blood vessels. We have shown previously that estrogen inhibits vascular injury response to the same extent in female wild-type (WT), ERα knockout (ERαKO_CH), and ERβ knockout (ERβKO_CH) mice. We generated mice harboring disruptions of both ERα and ERβ genes (ERα,βKO_CH) by breeding and studied the effect of 17β-estradiol (E2) on vascular injury responses in ovariectomized female ERα,βKO_CH mice and WT littermates. E2 inhibited increases in vascular medial area following injury in the WT mice but not in the ERα,βKO_CH mice, demonstrating for the first time that the two known estrogen receptors are necessary and sufficient to mediate estrogen inhibition of a component of the vascular injury response. Surprisingly, as in WT littermates, E2 still significantly increased uterine weight and inhibited vascular smooth muscle cell (VSMC) proliferation following injury in the ERα,βKO_CH mice. These data support that the role of estrogen receptors differs for specific components of the vascular injury response in the ERα,βKO_CH mice. The results leave unresolved whether E2 inhibition of VSMC proliferation in ERα,βKO_CH mice is caused by a receptor-independent mechanism, an unidentified receptor responsive to estrogen, or residual activity of the ERα splice variant reported previously in the parental ERαKO_CH mice. These possibilities may be resolved by studies of mice in which ERα has been fully disrupted (ERαKO_St), which are in progress.