The aim of this study was to investigate the suppressive activity of 70% ethanol extract and its dichloromethane fraction from Auricularia auricula-judae against adipogenesis and lipogenesis in 3T3-L1 preadipocytes. The ethanol extract and its dichloromethane fraction suppressed the differentiation and decreased lipid droplets in vitro. The dose-dependent increasing concentration of glycerol and lower triglycerides accumulation were found significantly (P < 0.05) with the treatment of both fractions from 70% ethanolic Auricularia auricula-judae extract and the glycerol-3-3phosphate dehydrogenase (GPDH) activity was also inhibited by both extracts. Further, the expression of adipogenic mRNAs were investigated by RT-PCR amplification. The key transcriptional factors, PPARγ and C/EBPα were decreased significantly at dose-dependent manner by both extracts of Auricularia auricula-judae. The expressions of LPL and FAS were also decreased by presence of these extracts. The decreased expressions of C/EBPβ, C/EBPγ and ACC1 were observed only by ethanol extract at 300 μg/ml concentration, while the expression of SREBP-1c, GLUT4 and aP2 were not altered in the 3T3-L1 adipocytes. These changes were occurred without the cytotoxic effect of both extracts against 3T3-L1 adipocytes in vitro. A positive control, fenofibrate inhibited the differentiation, triglycerides accumulation through PPARγ signaling by 2/3 reduction of PPARγ expression. Thus, these findings suggest that both extracts of Auricularia auricula-judae might be used to inhibit the differentiation of 3T3-L1 preadipocytes and reduction of triglycerides accumulation.