IL-10 is an immunomodulatory cytokine that plays an obligate role in preventing spontaneous enterocolitis in mice. However, little is known about IL-10 function in the human intestinal mucosa. We showed here that IL-10 was constitutively expressed and secreted by the human normal colonic mucosa, including epithelial cells. Depletion of IL-10 in mucosal explants induced both downregulation of the IL-10-inducible, immunosuppressive gene BCL3 and upregulation of IFN-gamma, TNF-alpha, and IL-17. Interestingly, TGF-beta blockade also strongly induced IFN-gamma production. In addition, the high levels of IFN-gamma produced upon IL-10 depletion were responsible for surface epithelium damage and crypt loss, mainly by apoptosis. Polymyxin B, used as a scavenger of endogenous LPS, abolished both IFN-gamma production and epithelial barrier disruption. Finally, adding a commensal bacteria strain to mucosa explant cultures depleted of both IL-10 and LPS reproduced the ability of endogenous LPS to induce IFN-gamma secretion. These findings demonstrate that IL-10 ablation leads to an endogenous IFN-gamma-mediated inflammatory response via LPS from commensal bacteria in the human colonic mucosa. We also found that both IL-10 and TGF-beta play crucial roles in maintaining human colonic mucosa homeostasis.