We previously reported the anti-obesity effects of immature persimmon ethanol extract by analyzing changes in body weight, visceral fat weight, blood biochemicals, and fat production-related gene expression levels in a high-fat diet-induced obese mouse model. Therefore, this study investigated whether the 50% ethanol extract of Diospyros kaki Thunb. regulated lipid metabolism by identifying its mechanism in 3T3-L1 cells. As a result, it did not influence cell viability or toxicity, and a significant decrease in lipid droplet accumulation was observed in a concentration-dependent manner during adipogenesis (adipogenic differentiation). Consistent with the animal studies, Diospyros kaki Thunb. extract (in a dose-dependent manner) suppressed peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding proteins, which induce adipocyte differentiation in 3T3-L1 preadipocytes. In addition, it increased the levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase in a dose-dependent manner in 3T3-L1 cells. Finally, we confirmed that sterol regulatory element-binding protein-1c is regulated by AMPK phosphorylation. These results indicated that immature persimmons (Diospyros kaki Thunb.) may serve as a natural agent for the prevention or treatment of obesity via the AMPK pathway.