Lactoferrin was reported to exert modulatory effects on lipid metabolism, but the regulatory mechanisms remain unclear. The present study investigated the beneficial effects of lactoferrin and their underlying mechanisms in high-fat diet-induced obese C57BL/6J mice. Oral administration of lactoferrin at 100 mg per body weight for 15 weeks significantly reduced weight gain, visceral adiposity, and serum glucose, leptin, and lipid levels in high-fat diet-induced obese mice. Hepatic steatosis in the obese mice was significantly improved. Expression of adipogenic and inflammation-related genes and proteins (SREBP-1c, FAS, MCP-1, leptin) was suppressed in the liver and epididymal adipose tissue of the obese mice. The present findings demonstrate that lactoferrin positively regulated lipid metabolism and improved hepatic steatosis in obese mice. The mechanisms of action for these effects may be attributed to suppression of lipogenic gene expression and amelioration of inflammation in the liver and epididymal adipose tissue.