Cacao extract (CE) consumption has beneficial effects on human health, such as lowering the risk of obesity. However, the underlying molecular mechanism for the anti-obesity effect of CE remains incompletely understood. Here, we used a 50% aqueous alcohol extract of cacao mass, which is rich in methylxanthine derivatives (about 11%) and poor in flavan-3-ols (less than 1%), and assessed the suppression effects of this extract on adipocyte differentiation to investigate the anti-obesity mechanism. CE dose-dependently decreased fat accumulation in 3T3-L1 cells without affecting cell viability. CE also dose-dependently decreased the protein and gene expression levels of two adipogenesis-related transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding proteins (C/EBPs). Moreover, CE decreased protein expression levels of sterol regulatory element-binding protein 1 (SREBP1) and its downstream fatty acid synthase (FAS), which was accompanied by the retained localization of SREBP1 in the cytoplasm of 3T3-L1 cells. After ICR mice were fed a diet containing 1% CE for 1 wk, their white adipose tissue weight was lower, whereas their brown adipose tissue weight was higher compared with those of control animals. Additionally, the protein expression levels of PPARγ, C/EBPs, SREBP1, and FAS in the white adipose tissue of these mice were also lower than those in control animals. In contrast, diet supplementation with CE induced higher levels of phosphorylated AMP-activated protein kinase (AMPK) and its downstream acetyl-CoA carboxylase. In conclusion, methylxanthine derivative-rich CE decreases fat accumulation in adipocytes by downregulating the expression of the adipocyte differentiation master regulators through the activation of AMPK.