BACKGROUND AND AIMS: Inflammatory bowel disease patients exhibit altered expression of nuclear estrogen receptors alpha and beta (ERalpha and ERbeta) and G-protein coupled estrogen receptor 1 (GPER1). We previously showed that deletion of ERalpha protects against intestinal damage selectively in female mice; however, the mechanisms conferring sex-specific protection are poorly understood. The goal of this study was to compare ERalpha- and ERbeta-specific mechanisms contributing to intestinal epithelial function in males and females. METHODS: Expression of ERalpha, ERbeta, and GPER1 was evaluated in colonocytes from wild-type male and female mice. Intestinal epithelial cell (IEC)-specific ERalpha and ERbeta knockout mice were developed and challenged with dextran sulfate sodium. Colonic organoids were used to identify estrogen-dependent and estrogen-independent effects on cellular growth, differentiation, and transcriptional regulation in wild-type, ERalpha-KO, and ERbeta-KO IECs. RESULTS: Colonic IECs showed significant expression of ERalpha, ERbeta, and GPER1 as well as Cyp19A1, which catalyzes production of 17beta-estradiol (estrogen). Female mice lacking ERalpha specifically in colonic IECs showed protection from dextran sulfate sodium-induced injury, whereas males showed increased pathology. Organoids derived from male ERalpha-KO mice showed enhanced proliferation and decreased expression of key functional genes even without exogenous estrogen; however, colonoids derived from female ERalpha-KO mice showed a protective gene signature. These findings reveal that deletion of ERalpha contributes to differential effects in male and female IECs, contributing to females' resistance to intestinal injury and inflammation. CONCLUSION: ERalpha signaling within IECs drives opposing sex-dependent effects on the development, regenerative capacity, and inflammatory susceptibility of the intestinal epithelium.