MUC2 mucin is the major glycoprotein in colonic mucus that separates intestinal microbiota from underlying host cells and serves as a food source for some eubacteria. MUC2 deficiency results in impaired epithelial barrier function, imbalance in gut microbiota and spontaneous colitis. Use of probiotics has been shown to have protective effect against colitis. In this study we tested whether the probiotic mixture VSL#3 required an intact mucin barrier to exert its beneficial effect using Muc2 mucin deficient (Muc2^-/-) and Muc2^+/+ littermates. VSL#3 treatment alone reduced basal colonic pro-inflammatory cytokine levels and improved epithelial barrier functions in Muc2^-/- animals. Likewise, in DSS-induced colitis, VSL#3 administration dampened the pro-inflammatory chemokines KC, MCP-1 and MIP-2 and up regulated tissue regeneration growth factors TGF-β, FGF-1 and VEGF-A that accelerated resolution of colitis symptoms in Muc2^-/- animals. Importantly, improved colonic health in VSL#3 treated animals was associated with attenuated reactive oxygen species (ROS) production by peritoneal macrophages, restoration of antimicrobial peptide gene expression in the small intestine and increased abundance of bacterial commensals in the gut. The beneficial effects of VSL#3 in Muc2^-/- animals were mediated by acetate, an important short chain fatty acid produced by gut bacteria. These studies provide evidence for the first time that VSL#3 can enhance epithelial barrier function by dampening pro-inflammatory cytokines and chemokine response, accelerating restitution and altering commensal microbiota in the absence of a functional mucus barrier.