The prevalence of obesity and type 2 diabetes in Japan is rising, largely due to the westernization of dietary habits. Controlling adipogenesis is a key strategy for prevention. Eicosapentaenoic acid (EPA), an n-3 polyunsaturated fatty acid, is known to exert anti-adipogenic effects; however, its detailed molecular mechanism during the critical “differentiation induction phase” remains unclear. This study aimed to elucidate how EPA suppresses adipogenesis in 3T3- L1 cells, with a focus on the cAMP signaling pathway. Cells were treated with EPA (50 μM) exclusively during the 48- hour differentiation induction phase. Lipid accumulation was evaluated by Oil Red O staining, and signaling pathways were analyzed using specific antagonists and Western blotting. EPA treatment markedly reduced lipid accumulation. Since antagonists of PPARα and PPARγ also inhibited adipogenesis, EPA is unlikely to exert its effects through activation of these receptors. Mechanistically, EPA decreased PKA phosphorylation, suggesting reduced intracellular cAMP levels. This key finding of cAMP depletion implies a concurrent shutdown of cAMP‑dependent pathways, including Epac, which is essential for differentiation. We propose that EPA exerts its anti-adipogenic effect primarily by suppressing cAMP production via a Gαi-protein-coupled receptor mechanism, thereby inhibiting the initiation of adipogenesis. Keywords: Adipogenesis, cAMP, Eicosapentaenoic acid, Gαi-coupled receptor, Signal transduction