Objective To explore the effects of 3'-hydroxygenistein on the proliferation and differentiation of 3T3-L1 preadipocytes and to elucidate the related mechanism. Methods 3T3-L1 preadipocytes were cultured and treated with different dosages of 3'-hydroxygenistein. Cell proliferation was analyzed by counting cell numbers and MTT assay; DNA synthesis of 3T3-L1 was investigated by bromodeoxyuridine (BrdU) incorporation assay; the degree of preadipocytes differentiation was evaluated by Oil red O staining; and the expressions of peroxisome proliferation activated receptor γ (PPARγ) and CAAT/enhancer binding protein (C/EBPα) were detected at mRNA and protein level by real-time PCR and Western blotting analysis, respectively. Results Pretreatment with 3'-hydroxygenistein (10-50 μmol/L) for 24, 48 and 72 h markedly promoted 3T3-L1 preadipocyte proliferation and differentiation in a dose-effect manner (P<0.05, P<0.01). It also significantly facilitated the DNA synthesis in 3T3-L1 preadipocytes in a dose-dependent manner (P<0.05). Similar to rosiglitazone (ROZ), 3'-hydroxygenistein (at 10 or 50 μmol/L) also resulted in more lipid droplets in 3T3-L1 preadipocytes (P<0.05, P<0.01). Furthermore, 3'-hydroxygenistein greatly increased the mRNA and protein expression of PPARγ and C/EBPα in 3T3-L1 preadipocytes (P<0.01). Conclusion 3'-hydroxygenistein can promote the proliferation and DNA synthesis of 3T3-L1 preadipocytes; it can also enhance the accumulation of lipid drops and increase the terminal differentiation of preadipocyts, which might be associated with its ability to increase the expression of PPARγ and C/EBPα.