Fucosterol is a sterol metabolite of brown algae and regulates genes involved with cholesterol homeostasis. As a part of our continuous search for anti-obesity agents from natural marine sources, the anti-adipogenic activities of Ecklonia stolonifera and its sterol, fucosterol, were evaluated for the inhibition of adipocyte differentiation and lipid formation. Oil Red O staining was used to evaluate triglyceride contents in 3T3-L1 pre-adipocytes primed by differentiation medium (DM) I and DM II. The methanolic extract of E. stolonifera showed strong anti-adipogenic activity, and was thus fractionated with several solvents. Among the tested fractions, the dichloromethane (CH2Cl2) fraction was found to be the most active fraction, with significant inhibition (40.5 %) of intracellular lipid accumulation at a non-toxic concentration, followed by the ethyl acetate fraction (30.2 %) at the same concentration, while the n-butanol and water fractions did not show inhibitory activity within the tested concentrations. The strong anti-adipogenic CH2Cl2-soluble fraction was further purified by a repeated chromatography to yield fucosterol. Fucosterol reduced lipid contents in a concentration-dependent manner without showing any cytotoxicity. Fucosterol treatment also yielded a decrease in the expression of the adipocyte marker proteins peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) in a concentration-dependent manner. Taken together, these results suggest that fucosterol inhibits expression of PPARγ and C/EBPα, resulting in a decrease of lipid accumulation in 3T3-L1 pre-adipocytes, indicating that the potential use of E. stolonifera and its bioactive fucosterol as an anti-obesity agent.