This study was to investigate the effect of iodine-enriched yolk extract on fat accumulation in vitro in cultured 3T3-L1 adipocyte for confirming the function as the functional food. The extracts of iodineenriched and ordinary yolk at the doses of 5, 10, 50 μg/mL were treated after adipocyte differentiation and their effects in intracellular fat accumulation were compared by the analyzing the oil-red staining, GPDH activity and adipogenic-associated genes. Iodine-enriched yolk with dose of 50 μg/mL significantly inhibited the fat accumulation in adipocytes compared with ordinary yolk. In addition, adipogenic-associated genes, i.e. PPAR-γ2, adipogenin, leptin and adiponectin, was significantly inhibited by treatment of iodine-enriched yolk. However, iodide ion (I-) treatment did not affect the fat accumulation in cultured adipocytes. These results indicate that the iodine bounded components including iodine peptide, not iodide ion, in iodine-enriched yolk may inhibit the fat accumulation in cultured adipocytes.