AMP-activated protein kinase (AMPK) is a central player in glucose and lipid metabolism while its role in adipocyte differentiation remains obscure. A-769662, a novel activator of AMPK, has been implicated to reduce body weight gain and decrease liver and plasma triglyceride levels via increasing fatty acid oxidation and reducing fatty acid synthesis in vivo. In this study, we examined the effects of A-769662 on adipocyte differentiation using 3T3-L1 cells. We found that A-769662 significantly inhibited 3T3-L1 differentiation via down-regulation of adipogenesis-related transcription factors and adipogenic markers. The inhibitory effect mainly occurred at the early phase of differentiation through inhibition of mitotic clonal expansion (MCE), which was essential for adipogenesis. A-769662 also decreased cell viability of differentiating and mature 3T3-L1 adipocytes. Moreover, treatment of differentiating 3T3-L1 cells with A-769662 resulted in AMPK over-activation, which led to an increased phosphorylation and inactivation of acetyl-CoA carboxylase (ACC), an important enzyme for the synthesis and usage of fatty acids. Taken together, these results suggest that A-769662 inhibits 3T3-L1 adipogenic differentiation in several ways and therefore may be a promising compound for the treatment of obesity.