Microbial dysbiosis and increased intestinal permeability is a target for prevention or reversal of weight gain in high-fat (HF) diet-induced obesity (DIO). Prebiotic milk oligosaccharides (MO) have been shown to benefit the host intestine, but have not been used in DIO. We hypothesized that supplementation with bovine MO would prevent the deleterious effect of HF diet on the gut microbiota and intestinal permeability, and attenuate development of the obese phenotype. C57BL/6 mice were fed a control diet (LF), HF (40% fat/kcal), or HF + prebiotic (6%/Kg BMO or inulin) for 1, 3 or 6 weeks. Gut microbiota and intestinal permeability were assessed in the ileum, cecum and colon. Addition of BMO to the HF diet significantly attenuated weight gain, decreased adiposity and decreased caloric intake; inulin supplementation also lowered weight gain and adiposity, but this did not reach significance. BMO and inulin completely abolished the HF diet-induced increase in paracellular and transcellular permeability in the small and large intestine. Both BMO and inulin increased abundance of beneficial microbes Bifidobacterium and Lactobacillus in the ileum. However, inulin supplementation altered phylogenetic diversity and decreased species richness. We conclude that addition of BMO to the HF diet completely prevented increases in intestinal permeability and microbial dysbiosis and was partially effective to prevent weight gain in DIO.