Objective：To investigate the inhibitory effects of obestatin on proliferation and differentiation of 3T3-L1 preadipocytes. Methods: Obestatin (10-8 mmol/L, 10-9 mmol/ L, 10-10 mmol/ L, 10-11 mmol/ L,and 10-12 mmol/L) and ghrelin (10-10 mmol/L) were used to treat 3T3-L1 preadipocytes. Cell proliferation was assessed by MTT assay and the results were compared with that of blank control group. The differentiation of 3T3-L1 preadipocytes (from day 1 to day 10) was interfered with obestatin or ghrelin (both at 10-10 mmol/L). Intracellular fat accumulation in differentiated adipocytes was determined by oil red O staining and the expression of PPARγ2 mRNA was detected by RT-PCR; the results were compared with that of control group (induced with routine inducer). Results: Compared with the blank control group, obestatin-treated groups (various concentrations of obestatin) had significantly less cells(P<0.05). Oil red O staining revealed that, compared with control group, the formation of lipid droplets was significantly decreased after 10 days’ of treatment with 10-10mmol/L obestatin (P<0.05). The expression of PPARγ2 gene increased with the progress of 3T3-L1 preadipocytes differentiation. PPARγ2 mRNA level in mature adipocytes of obestatin group was significantly lower than that in the cells of control group. The effect of ghrelin was contrary to that of obestatin. Conclusion: Obestatin can inhibit the proliferation and differentiation of 3T3-L1 preadipocyes.