Hyperhomocysteinemia (HHcy) is a characteristic metabolic abnormality in several pathological conditions, including hypertension, diabetes and alcoholic liver disease. Emerging evidence indicates that adipose tissue contributes to HHcy and homocysteine (Hcy) conversely affects adipose tissue function. However, the specific effect of Hcyon adipogenesis is poorly understood. In the present study, we investigated the effects and mechanisms of Hcy on adipogenic process using 3T3-L1 preadipocytes, a well-established in vitro model for the study of adipogenesis. Confluent mouse embryo 3T3-L1 preadipocytes (D0) were exposed to differentiation cocktail for three days (D3). Then, cells were transferred to insulin-containing medium and re-fed every two days. Maturation of adipocytes was confirmed by Oil Red O staining of lipid droplets on day 7. Exogenous Hcy was added to the culture medium on either D0 or D3. At day 7, adipogenesis indices were measured. Our data indicated that both Hcy addition protocols suppressed adipogenic process, evidenced by decreased lipid accumulation and downregulated gene expressions of adipocyte protein 2 and peroxisome proliferator-activated receptor gamma (PPAR-gamma), implying that Hcy exerted inhibitory effects on both mitotic clonal expansion (MCE) stage and differentiation stage. Further study showed that Hcy suppresses MCE via decreasing retinoblastoma protein phosphorylation and E2F-1 protein expression. To delineate the critical involvement of PPAR-gamma in Hcy-induced suppression on adipogenesis, we employed rosiglitazone, a specific PPAR-gamma agonist, to replace insulin for the inductive stimulus of adipogenesis. Our results showed that Hcy suppressed rosiglitazone-induced adipogenesis in a similar fashion as this by insulin, suggesting that inhibition of PPAR-gamma transactivation was critically involved in the Hcy-induced inhibitory effect on adipogenesis. Taken together, our data indicate that Hcy suppressed adipogenesis in 3T3-L1 preadipocytes and the inhibition of PPAR-gamma transactivity may, at least partially, contribute to the suppressive effect.