The study investigates the role of Claudin-7 (Cldn-7) in maintaining intestinal epithelial homeostasis, focusing on its impact on colonic Lgr5+ stem cells. Previous research indicated that the absence of Cldn-7 in intestinal epithelial cells leads to spontaneous colitis. In this study, IEC- and ISC-specific Cldn-7 knockout mice were used to explore Cldn-7’s regulatory effects on colonic Lgr5+ stem cells during both physiological and inflammatory conditions. Results show that Cldn-7 deletion disrupts the self-renewal and differentiation of colonic stem cells, as well as colonic organoid formation in vitro. Moreover, Cldn-7 knockout models displayed increased susceptibility to experimental colitis, impaired epithelial repair and regeneration, and enhanced differentiation towards the secretory lineage. Mechanistically, the study reveals that Cldn-7 facilitates the proliferation, differentiation, and organoid formation of Lgr5+ stem cells by maintaining Wnt and Notch signaling pathways in the colonic epithelium. Overall, the findings provide new insights into ISC function and colonic epithelial homeostasis maintenance.