The purpose of this study was to investigate the antiobesity effect of Sanguisorba officinalis L. (SOL) in 3T3-L1 adipocytes and obese C57BL/6J mice. SOL was extracted with water and 30%, 50%, 70%, and 100% ethanol (EtOH). 3T3-L1 adipocytes were treated with SOL extracts (100 μg/mL) during the differentiation period. Triglyceride (TG) accumulation was determined by Oil Red O staining, and the expression of adipocyte-specific proteins was measured by Western blot analysis. C57BL/6J mice were fed a high-fat diet to induce obesity and were orally administered SOL 50% ethanol extract (50, 100, and 200 mg/kg) for 8 weeks. Among the SOL extracts, the 50% EtOH extract considerably inhibited TG accumulation through the downregulation of PPARγ, C/EBPα, and SREBP-1c in 3T3-L1 adipocytes. In addition, the 50% ethanol extract reduced body weight and adipose tissue weight and improved serum lipid profiles through downregulation of PPARγ, C/EBPα, FABP4, and ACC and upregulation of adiponectin and CPT-1 in obese C57BL/6J mice fed a high-fat diet. These results suggested that the SOL 50% EtOH extract may have an antiobesity effect through the regulation of transcription factors related to adipogenesis, lipogenesis, and lipolysis.