Limited hydrolysis of isolated soy protein (ISP) with Neutrase for 4 h to obtain the hydrolysate (NH4h) revealed the ability to suppress glycerol-3-phosphate dehydrogenase (GPDH) activity and relative lipid accumulation (RLA) in 3T3-L1 cells during differentiation. Lower GPDH activity or RLA indicates higher anti-adipogenic activity. Sequentially fractionating NH4h with 30-1 kDa (kilo-daltons) molecular weight cut-off (MWCO) membranes to obtain the 1 kDa concentrate resulted in further enhancing its anti-adipogenic activity in the cells. The GPDH activity significantly decreased from 280 to 100 U/mg protein (p < 0.05). When comparing the high-performance size-exclusion chromatography (HPSEC) profiles, the most active peptide for the anti-adipogenic activity was primarily composed of peptides with molecular weight between 1300 and 2200 Da. The in vitro effect of gastrointestinal protease on the anti-adipogenic activity of I kDa concentrate was also investigated. The results suggested that gastrointestinal proteases have very little effect on anti-adipogenic activity of the concentrate. According to the Western immunoblot analysis, I kDa concentrate inhibits adipogenesis by affecting the expression of peroxisome proliferators-activated receptor-gamma (PPAR-gamma) and the CCAATIenhancer binding protein alpha (C/EBP alpha) during 3T3-L1 cells differentiation. (C) 2009 Elsevier Ltd. All rights reserved.