Mori Folium, the leaf of Morus alba, is a traditional medicinal herb that shows various pharmacological activities such as antiinflammatory, antidiabetic, antimelanogenesis, antioxidant, antibacterial, antiallergic, and immunomodulatory activities. However, the mechanisms of their inhibitory effects on adipocyte differentiation and adipogenesis remain poorly understood. In the present study, we investigated the inhibition of adipocyte differentiation and adipogenesis by ethanol extracts of Mori Folium (EEMF) in 3T3-L1 preadipocytes. Treatment with EEMF suppressed the terminal differentiation of 3T3-L1 preadipocytes in a dose-dependent manner, as confirmed by a decrease in the lipid droplet number and lipid content through Oil Red O staining. EEMF significantly reduced the accumulation of cellular triglyceride, which is associated with a significant inhibition of pro-adipogenic transcription factors, including sterol regulatory element-binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor-γ (PPARγ), and CCAAT/enhancer-binding proteins α (C/EBPα) and β (C/EBPβ).In addition, EEMF potentially downregulated the expression of adipocyte-specific genes, including adipocyte fatty acid binding protein (aP2) and leptin. Furthermore, EEMF treatment effectively increased the phosphorylation of the AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase (ACC); however, treatment with a potent inhibitor of AMPK, compound C, significantly restored the EEMF-induced inhibition of pro-adipogenic transcription factors and adipocyte-specific genes. These results together indicate that EEMF has preeminent effects on the inhibition of adipogenesis through the AMPK signaling pathway, and further studies will be needed to identify the active compounds in Mori Folium.