We investigated the bacteriocin plantaricin EF (PlnEF) system for its contributions to mediated benefits in a mouse model of diet-induced obesity. C57BL/6J mice on a high-fat diet (HFD) were administered a rifampicin resistant mutant of NCMIB8826 (NICMB8826-R) or an isogenic Δ mutant strain, LM0419, every 48 h for nine weeks. Mice fed wild-type , but not LM0419, reduced their consumption of the HFD starting three weeks into the study and exhibited an overall 10% reduction in weight gain. The responses were independent of glucose homeostasis, as both NCMIB8826-R and LM0419 fed mice had improved oral glucose tolerance compared to sham controls. Although bacteriocins have antibacterial properties, the ileal, cecal, and fecal microbiota and cecocolic metabolomes were unchanged between mice fed either wild-type or the Δ mutant. Instead, only mice fed NCMIB8826-R showed an increased production of ZO-1 in ileal tissues. To verify a potential role for the plantaricin EF system in supporting intestinal epithelial function, synthesized PlnEF peptides were applied to Caco-2 cell monolayers challenged with TNF-α and IFN-γ. The combination of PlnE and PlnF were required to prevent sustained cytokine-induced losses to Caco-2 cell para- and transcellular permeability and elevated IL-8 levels. In conclusion, this study shows that probiotic ameliorates the effects of obesogenic diets through a mechanism that involves the plantaricin EF system and likely includes induced fortification of the intestinal epithelium.