Fatty acid synthase (FAS) is uniquely expressed at high levels in cancer cells and adipose tissue. The objectives of this study were to identify, purify and validate soy FAS inhibitory peptides and to predict their binding modes. Soy peptides were isolated from hydrolysates of purified β-conglycinin by co-immunoprecipitation and identified using LC-MS/MS. Three peptides, KNPQLR, EITPEKNPQLR and RKQEEDEDEEQQRE, inhibited FAS. The biological activity of these peptides was confirmed by their inhibitory activity against purified chicken FAS (IC50 = 79, 27 and 16 μm, respectively) and a high correlation (r = -0.7) with lipid accumulation in 3T3-L1 adipocytes. The FAS inhibitory potency of soy peptides also correlated with their molecular mass, pI value and the number of negatively charged and hydrophilic residues. Molecular modeling predicted that the large FAS inhibitory peptides (EITPEKNPQLR and RKQEEDEDEEQQRE) bond to the thioesterase domain of human FAS with lower interaction energies (-442 and -353 kcal·mol-1, respectively) than classical thioesterase inhibitors (Orlistat, -91 kcal·mol -1 and C75, -51 kcal·mol-1). Docking studies suggested that soy peptides blocked the active site through interactions within the catalytic triad, the interface cavity and the hydrophobic groove in the human FAS thioesterase domain. FAS thioesterase inhibitory activities displayed by the synthetic soy peptides EITPEKNPQLR and RKQEEDEDEEQQRE (IC50 = 10.1 ± 1.6 and 10.7 ± 4.4 μm, respectively) were higher than C75 (58.7 μm) but lower than Orlistat (0.9 μm). This is the first study to identify FAS inhibitory peptides from purified β-conglycinin hydrolysates and predict their binding modes at the molecular level, leading to their possible use as nutraceuticals. © 2010 FEBS.