Diphlorethohydroxycarmalol (DPHC) is one of the most abundant bioactive compounds in . The previous study suggested that DPHC possesses strong in vitro anti-obesity activity in 3T3-L1 cells. However, the in vivo anti-obesity effect of DPHC has not been determined. The current study explored the effect of DPHC on high-fat diet (HFD)-induced obesity in C57BL/6J mice. The results indicated that oral administration of DPHC (25 and 50 mg/kg/day for six weeks) significantly and dose-dependently reduced HFD-induced adiposity and body weight gain. DPHC not only decreased the levels of triglyceride, low-density lipoprotein cholesterol, leptin, and aspartate transaminase but also increased the level of high-density lipoprotein cholesterol in the serum of HFD mice. In addition, DPHC significantly reduced hepatic lipid accumulation by reduction of expression levels of the critical enzymes for lipogenesis including SREBP-1c, FABP4, and FAS. Furthermore, DPHC remarkably reduced the adipocyte size, as well as decreased the expression levels of key adipogenic-specific proteins and lipogenic enzymes including PPARγ, C/EBPα, SREBP-1c, FABP4, and FAS, which regulate the lipid metabolism in the epididymal adipose tissue (EAT). Further studies demonstrated that DPHC significantly stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in both liver and EAT. These results demonstrated that DPHC effectively prevented HFD-induced obesity and suggested that DPHC could be used as a potential therapeutic agent for attenuating obesity and obesity-related diseases.