Defects in gut barrier function are implicated in gastrointestinal (GI) disorders like inflammatory bowel disease (IBD), as well as in systemic inflammation. With the increasing incidence of IBD worldwide, more attention should be paid to dietary interventions and therapeutics with the potential to boost the natural defense mechanisms of gut epithelial cells. The current study aimed to investigate the protective effects of ATCC PTA 4659 in a colitis mouse model and delineate the mechanisms behind it. Wild-type mice were allocated to the control group; or given 3% dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis; or administered for 7 days as pretreatment; or for 14 days starting 7 days before subjecting to the DSS. Peroral treatment with improved colitis severity clinically and morphologically and reduced the colonic levels of Tumor necrosis factor-α (TNF-α) (), Interleukin 1-β (), and nterferon-γ (), the crucial pro-inflammatory cytokines in colitis onset. It also prevented the CD11bLy6G neutrophil recruitment and the skewed immune responses in mesenteric lymph nodes (MLNs) of CD11bCD11c dendritic cell (DC) expansion and Foxp3CD4 T-cell reduction. Using 16S rRNA gene amplicon sequencing and RT-qPCR, we demonstrated a colitis-driven bacterial translocation to MLNs and gut microbiota dysbiosis that were in part counterbalanced by treatment. Moreover, the expression of barrier-preserving tight junction (TJ) proteins and cytoprotective heat shock protein (HSP) 70 and HSP25 was reduced by colitis but boosted by treatment. A shift in expression pattern was also observed with HSP70 in response to the pretreatment and with HSP25 in response to -DSS. In addition, the changes of HSPs were found to be correlated to bacterial load and epithelial cell proliferation. In conclusion, our results demonstrate that the human-derived strain 4659 confers protection in experimental colitis in young mice, while intestinal HSPs may mediate the probiotic effects by providing a supportive protein-protein network for the epithelium in health and colitis.