We applied licorice flavonoid oil (LFO) to high-fat diet-induced obese C57BL/6J mice and investigated its effect. LFO contains hydrophobic flavonoids obtained from licorice by extraction with ethanol. The oil is a mixture of medium-chain triglycerides, having glabridin, a major flavonoid of licorice, concentrated to 1.2% (w/w). Obese mice were fed on a high-fat diet containing LFO at 0 (control), 0.5%, 1.0%, or 2.0% for 8 weeks. Compared with mice in the control group, those in the 1% and 2% LFO groups efficiently reduced the weight of abdominal white adipose tissues and body weight gain. A histological examination revealed that the adipocytes became smaller and the fatty degenerative state of the hepatocytes was improved in the 2% LFO group. A DNA microarray analysis of the liver showed up-regulation of those genes for beta-oxidation and down-regulation of those for fatty acid synthesis in the 2% LFO group. These findings suggest that LFO prevented and ameliorated diet-induced obesity via the regulation of lipid metabolism-related gene expression in the liver.