In a previous study, we demonstrated the anti-obesity and hypolipidemic effects of Boussingaulti gracilis Miers var. pseudobaselloides Bailey in high-fat diet-induced obese rats. The present study investigated the molecular mechanisms by which B. gracilis Miers var. pseudobaselloides Bailey ethanol extract (BGE) conferred antidifferentiation and anti-adipogenic effects in the 3T3-L1 preadipocyte differentiation model. BGE treatment significantly and dose-dependently suppressed lipid accumulation and down-regulated the expression of major transcription factors involved in adipogenesis, such as peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein α, sterol regulatory element-binding proteins, and their target genes. It is important that treatment with BGE increased phosphorylation of AMP-activated protein kinase (AMPK), which is one of the rate-limiting enzymes in the fatty acid synthesis pathway, and its direct downstream protein, acetyl-coenzyme A carboxylase. These results suggest that BGE may exert anti-adipogenic effects through regulation of AMPK activity and expression of genes involved in lipogenesis.