Estrogen is atheroprotective and a high-affinity ligand for both known estrogen receptors, ERα and ERβ. However, the role of the ERα in early-stage atherosclerosis has not been directly investigated and is incompletely understood. ERα-deficient (ERα−/−) and wild-type (ERα+/+) female mice consuming an atherogenic diet were studied concurrent with estrogen replacement to distinguish the actions of 17β-estradiol (E2) from those of ERα on the development of early atherosclerotic lesions. Mice were ovariectomized and implanted with subcutaneous slow-release pellets designed to deliver 6 or 8 μg/day of exogenous 17β-estradiol (E2) for a period of up to 4 months. Ovariectomized mice (OVX) with placebo pellets (E2-deficient controls) were compared to mice with endogenous E2 (intact ovaries) and exogenous E2. Aortas were analyzed for lesion area, number, and distribution. Lipid and hormone levels were also determined. Compared to OVX, early lesion development was significantly (p < 0.001) attenuated by E2 with 55–64% reduction in lesion area by endogenous E2 and >90% reduction by exogenous E2. Compared to OVX, a decline in lesion number (2- to 4-fold) and lesser predilection (~4-fold) of lesion formation in the proximal aorta also occurred with E2. Lesion size, development, number, and distribution inversely correlated with circulating plasma E2 levels. However, atheroprotection was independent of ERα status, and E2 athero-protection in both genotypes was not explained by changes in plasma lipid levels (total cholesterol, triglyceride, and high-density lipoprotein cholesterol). The ERα is not essential for endogenous/exogenous E2-mediated protection against early-stage atherosclerosis. These observations have potentially significant implications for understanding the molecular and cellular mechanisms and timing of estrogen action in different estrogen receptor (ER) deletion murine models of atherosclerosis, as well as implications to human studies of ER polymorphisms and lipid metabolism. Our findings may contribute to future improved clinical decision-making concerning the use of hormone therapy.