(−)-Ternatin, a highly N-methylated cyclic peptide, inhibits fat accumulation in 3T3-L1 cells and reduces fat mass in mice. However, the mechanism for its anti-adipogenic effect has remained unknown. To examine the mechanism used by (−)-ternatin to inhibit adipocyte differentiation, we examined the effects of (−)-ternatin and [l-Ala4]ternatin, an inactive analog of (−)-ternatin, on the expression of adipocyte markers and lipogenic enzymes. We found that (−)-ternatin potently reduced mRNA expression of several adipocyte markers in a dose-dependent manner, whereas [l-Ala4]ternatin showed no effects. At the immediate early phase, (−)-ternatin, but not [l-Ala4]ternatin, reduced the expression of Srebp1c, Fas, Acc2 and C/EBP-α while showing no effects on C/EBP-β and C/EBP-δ. These results suggest that (−)-ternatin affects the mid-to late differentiation stages of adipocytes. Consistent with the decreased expression of lipogenic enzymes, (−)-ternatin potently inhibited triglyceride synthesis. Intriguingly, (−)-ternatin also inhibited triglyceride synthesis in rat primary hepatocytes, suggesting that the potential action sites for (−)-ternatin are shared by adipocytes and liver. Although the target molecule of (−)-ternatin remains unknown, our data suggest that (−)-ternatin and its potential target might provide a new therapeutic approach to metabolic disorders.