Probiotics can play a role in enhancing intestinal barrier function. However, the underlying mechanisms are not fully understood. The aim of this study was to examine the effects of VSL#3 probiotics on colonic epithelium permeability, tight junction protein expression and MAPKs signaling pathways in vivo and in vitro. In vivo, acute colitis was induced by administration of 3.5% dextran sodium sulfate for 7 days. Rats in two groups were treated with either 15 mg VSL#3 or placebo via a gastric tube once daily after induction of colitis. Tight junction protein expression and the MAPKs signaling pathways were studied by immunohistochemistry and immunoblotting. In vitro, HT-29 cells were exposed to TNF-α for up to 48 h with or without pre-treatment with a p38 MAPK inhibitor, an ERK inhibitor or a JNK inhibitor. Then tight junction proteins and the phosphorylation of MAPKs were examined in the presence or absence of VSL#3. In vivo, VSL#3 probiotics significantly ameliorated the disease activity index from Day 4 onward. In acute colitis rats, decreased expression of the tight junction proteins were observed, whereas VSL#3 therapy prevented these changes and increased the expression of phosphorylated p38 (P-p38), and of phosphorylated ERK (P-ERK). In vitro, tight junction proteins, P-p38 and P-ERK in the VSL#3 group were significantly higher than in the control and TNF-α groups. The p38 MAPK inhibitor and the ERK inhibitor could effectively prevent this effect. VSL#3 probiotics protected the epithelial barrier and increased the tight junction protein expression in vivo and in vitro by activating the p38 and ERK signaling pathways.