The role of AMP-activated protein kinase (AMPK) in adipocyte differentiation is not completely understood. Here we reported that an AMPK inhibitor, compound C, significantly inhibited adipogenic differentiation of 3T3-L1 cells in a dose dependent manner, and this inhibitory effect was primarily effective in the initial stage of differentiation. Compound C prevented the mitotic clonal expansion (MCE) of preadipocytes, probably by inhibiting expression of CCAAT/enhancer-binding protein (C/EBP)beta and delta, and subsequently blocked the expression of C/EBPalpha and peroxisome proliferator-activated receptor (PPAR)gamma and transcriptional activation of genes that produce the adipocyte phenotype. AMPK activity was also suppressed by compound C treatment during the early phase of adipogenic differentiation, which indicated that suppressed activation of AMPK by compound C may inhibit the MCE process of preadipocytes. Our results suggest that compound C might serve as a useful molecule in both basic and clinical research on adipogenesis and as a potential lead compound for the treatment of obesity.