Abstract Estrogen replacement therapy in postmenopausal women is associated with a reduction in colorectal cancer risk, potentially via interactions between 17β-estradiol (E2) and the estrogen receptors (ER) α and β. To study the role of E2 in intestinal tumor inhibition, we separately crossed C57BL/6J-Min/+ (Min/+) mice with Erα+/− and Erβ+/− mice to generate ER-deficient Min/+ progeny. We found an increased incidence of visible colon tumors and dysplastic microadenomas in ER-deficient Min/+ relative to Er+/+Min/+ controls. Small intestinal tumor numbers were unaffected. Invasive carcinomas were found only in Erα+/−Min/+ mice, suggesting that ERα plays additional non–cell autonomous roles that limit tumor progression. Histologic analyses of ER-deficient Min/+ colons, as well as colons from ovariectomized Min/+ mice (OvxMin/+) and E2-treated OvxMin/+ mice (OvxMin/+ +E2), revealed significant differences in crypt architecture, enterocyte proliferation, and goblet cell differentiation relative to Min/+ and Er+/+Apc+/+ (wild-type) controls. The expression of ERα and ERβ was regionally compartmentalized along the colonic crypt axis, suggesting functional antagonism. Our results indicate that ERα and ERβ are inhibitory modifiers of Apc-dependent colon tumorigenesis. As a result, loss of E2 and ER signaling in postmenopausal women may contribute to colorectal cancer development. [Cancer Res 2007;67(5):2366–72]