Our study investigated that the anti-obesity effect of the Trapa japonica Flerov extract (TJ) in differentiated 3T3-L1 adipocytes. To this end, 3T3-L1 cells were treated with TJ during their differentiation period. On the last day of the cell culture, we tested intracellular cAMP, FA, glycerol release, TG, and performed Oil Red O staining and Western blot assays. On the part of adipogenesis, lipogenesis, and lipolysis mechanism, TJ increased the cAMP (maximum 125.4%) levels and glycerol release (maximum four times) and decreased FA (maximum 35.1%) and TG (maximum 35.7%) levels. Furthermore, the protein expression levels of each mechanism-related factor were regulated in a dose-dependent manner. These results indicate that TJ reduced lipid accumulation by max 53.6% and 47.9%, respectively, in adipogenesis and lipolysis mechanisms. We expect this effect of TJ to be due to its component, ellagic acid. In conclusion, we found that TJ inhibits TG synthesis during adipogenesis and lipogenesis, promotes lipolysis, and thus, indicating its potential as a functional food for obesity prevention.