BACKGROUND/OBJECTIVES: Adipogenesis is part of the cell differentiation process in which undifferentiated fibroblasts (pre-adipocytes) become mature adipocytes with the accumulation of lipid droplets and subsequent cell morphological changes. Several transcription factors and food components have been suggested to be involved in adipogenesis. The aim of this study was to determine whether mulberry leaf ethanol extract (MLEE) affects adipogenesis in 3T3-L1 adipocytes. MATERIALS/METHODS: The 3T3-L1 adipocytes were treated with different doses of MLEE for 8 days starting 2 days post-confluence. Cell viability, fat accumulation, and adipogenesis-related factors including CCAAT-enhancer-binding protein alpha ($C/EBP{alpha}$), peroxisome proliferator-activated receptor gamma ($PPAR{gamma}$), $PPAR{gamma}$ coactivator 1 alpha (PGC-$1{alpha}$), fatty acid synthase (FAS), and adiponectin were analyzed. RESULTS: Results showed that MLEE treatments at 10, 25, 50, and $100{mu}g/ml$ had no effect on cell morphology and viability. Without evident toxicity, all MLEE treated cells had lower fat accumulation compared with control as shown by lower absorbances of Oil Red O stain. MLEE at 50 and $100{mu}g/ml$ significantly reduced protein levels of $PPAR{gamma}$, PGC-$1{alpha}$, FAS, and adiponectin in differentiated adipocytes. Furthermore, protein level of $C/EBP{alpha}$ was significantly decreased by the treatment of $100{mu}g/ml$ MLEE. CONCLUSION: These results demonstrate that MLEE treatment has an anti-adipogenic effect in differentiated adipocytes without toxicity, suggesting its potential as an anti-obesity therapeutic.