Gut microbiota has been suggested to participate in the pathogenesis of low-grade inflammation and obesity; bovine lactoferrin (bLF) as a dietary supplement is well known for its anti-microbial and prebiotic potency. However, the role of bLF in obesity as a prebiotic compound has not been investigated previously. We report that oral administration of 100 mg/kg BW bLF for 12 weeks decreased body weight gain and fat storage, reduced the blood cholesterol and glucose concentrations, and alleviated liver steatosis. The overexpression of leptin and plasminogen activator inhibitor (PAI-I) was suppressed, which could be attributed to decreased lipopolysaccharide (LPS) translocation and gut inflammation by bLF through positive modulation on gut microbiota, i.e. increasing the number of Bifidobacterium spp. and modestly inhibiting the growth of Enterobacteriales. Our results demonstrated that oral administration with bLF positively modulated gut microbiota, reduced inflammation and regulated lipid and glucose metabolism in high-fat diet induced obese mice.