The purpose of this study was to investigate the antiobesity effect and the mechanism of action of diphlorethohydroxycarmalol (DPHC) isolated from Ishige okamurae in 3T3-L1 cells. The antiobesity effects were examined by evaluating intracellular fat accumulation in Oil Red O-stained adipocytes. Based on the results, DPHC dose-dependently inhibited the lipid accumulation in 3T3-L1 adipocytes. DPHC significantly inhibited adipocyte-specific proteins such as SREBP-1c, PPARγ, C/EBP α, and adiponectin, as well as adipogenic enzymes, including perilipin, FAS, FABP4, and leptin in adipocytes. These results indicated that DPHC primarily acts by regulating adipogenic-specific proteins through inhibiting fat accumulation and fatty acid synthesis in adipocytes. DPHC treatment significantly increased both AMPK and ACC phosphorylation in adipocytes. These results indicate that DPHC inhibits the fat accumulation by activating AMPK and ACC in 3T3-L1 cells. Taken together, these results suggest that DPHC can be used as a potential therapeutic agent against obesity.