Garlic and its organosulfur compounds display hypolipidemic effects by inhibiting fatty acid and cholesterol synthesis. We investigated the anti-adipogenic effect of diallyl trisulfide (DATS), the second most abundant organosulfide in garlic oil. We studied the effects of DATS in 3T3-L1 preadipocytes and the mechanism involved in its action. DATS (0-75 μM) inhibited CCAAT/enhancer-binding protein (C/EBP) α and β and peroxisome proliferator-activated receptor (PPAR) γ mRNA and protein levels in a dose- and time-dependent manner, leading to a decrease of fatty acid synthase (FAS) expression and lipid accumulation in 3T3-L1 cells. Insulin treatment induced a transient increase in ERK phosphorylation in 3T3-L1 preadipocytes, and maximal induction was observed at 5 min and then declined. DATS, however, sustained ERK phosphorylation up to 120 min. In the presence of PD98059, the phosphorylation of ERK and suppression of PPARγ expression and DNA binding activity of PPARγ by DATS were reversed, and lipid accumulation was restored. In conclusion, these results indicate that DATS inhibits the differentiation of 3T3-L1 preadipocytes into adipocytes. DATS is likely to act by prolonging ERK activation, which leads to the down-regulation of adipogenic transcription factor expression during adipogenesis. Our results suggest that garlic may have potential as an anti-obesity agent.