Genistein, an isoflavone, was shown to have therapeutic effects for obesity, diabetes and cardiovascular diseases. This study investigated the effect and underlying mechanism of genistein on adipogenesis in 3T3-L1 preadipocytes. Genistein inhibited lipid accumulation and decreased the nonesterified fatty acid (NEFA) content of 3T3-L1 on day 6 after the induction of differentiation with methylisobutylxanthine, dexamethasone and insulin (MDI). Genistein recovered nitric oxide (NO) release suppressed by MDI and the results were consistent with the expression of endothelial NO synthase (eNOS) assayed by western blotting. Pretreatment with genistein inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) stimulated with 10 microg/mL of insulin. Furthermore, genistein inhibited the expression of fatty acid synthase (FAS) from 178% of the MDI group to 74%. SB203580, a p38 inhibitor, mimicked the FAS inhibition effect of genistein, suggesting that the inhibitory effect of genistein on FAS was partially via the p38 pathway. On the other hand, genistein abolished the phosphorylation of janus-activated kinase 2 (JAK2) in response to MDI. AG490, a JAK2 inhibitor, suppressed the expression of CCAAT/enhancer binding protein alpha (C/EBPalpha), a marker of adipocyte differentiation. The findings suggest that genistein attenuates the differentiation of 3T3-L1 involving multiple signal pathways.