P-Glycoprotein dysregulation and microbial imbalance have been implicated in inflammatory bowel diseases. Here we found that oral dosing of Mori Cortex, the root bark of Morus alba L. markedly alleviated inflammatory responses, reinstated microbial balance, and enhanced P-glycoprotein (P-gp) expression in rat colitis (UC) induced by oral administration of dextran sulfate sodium. The effects of Mori Cortex extract (MCE) on colon P-gp were examined using Caco-2 cells which revealed a time-dependent regulatory profile. The distinct effects on P-gp by individual main components may account for the direct biphasic effects of MCE. The involvement of gut microbiota in P-gp regulation by MCE was assessed by incubating the culture supernatant (CS) of fecal bacteria from normal, UC or MCE pretreated rats with Caco-2 cells. Interestingly, compared to normal CS, UC CS but not MCE CS diminished P-gp expression in Caco-2 cells, and this down-regulation could be reversed by pretreatment of Caco-2 cells with MCE. Moreover, MCE CS treated Caco-2 cells generated proinflammatory IL-1b and IL-8 comparable to that of the normal group and lower than the UC group, whereas, the anti-inflammatory IL-10 stimulated by MCE CS was significantly higher than the UC CS. In conclusion, MCE alleviated colitis-like symptoms and enhanced the intestinal epithelial integrity (P-gp up-regulation) in experimental colitis. The mechanisms involve both a direct effect and a gut microbiota-mediated pathway.