Bifidobacteria are important mediators of immune system development within the gastrointestinal system and immunological homeostasis. The present study explored the anti-colitic activity of H3-R2 in a murine dextran sulfate sodium (DSS)-induced model of ulcerative colitis (UC). Moreover, this study offers novel insight regarding the molecular basis for the probiotic properties of H3-R2 by analyzing the underlying mechanisms whereby H3-R2-derived proteins affect the intestinal barrier. H3-R2 administration was sufficient to alleviate clinical manifestations consistent with DSS-induced colitis, restoring aberrant inflammatory cytokine production, enhancing tight junction protein expression, and positively impacting overall intestinal microecological homeostasis in these animals. Moreover, the bifidobacteria-derived GroEL and transaldolase (TAL) proteins were found to regulate tight junction protein expression via the NF-κB, myosin light chain kinase (MLCK), RhoA/Rho-associated protein kinase (ROCK), and mitogen-activated protein kinase (MAPK) signaling pathways, preventing the lipopolysaccharide (LPS)-mediated disruption of the intestinal epithelial cell barrier.