The dried seeds of Iris lactea Pall. var. chinensis (Fisch.) Koidz, an important traditional Chinese medicine, are regarded to have effects of clearing heat, eliminating dampness and pharyngitis and so on. It has been used in the treatment of jaundice, diarrhea, leucorrhea and carbuncles. Previous phytochemical studies of Iris species showed the presence of flavones, isoflavones, triterpenes and stilbenes. In our previous research, we isolated five known oligostilbenes, vitisin A, vitisin B, vitisin C, vitisin D, and cis-vitisin A were successfully isolated from Iris lactea for the first time. The aim of this study was to assess the effects of these oligostilbenes on the differentiation and adipogenes in 3T3-L1 cells. Our results showed that vitisin A, vitisin B, cis-vitisin A significantly inhibited adipocytes differentiation and reduced lipid accumulation in 3T3-L1 cells. In addition, vitisin A, vitisin B, cis-vitisin A strongly suppressed the expression levels of adipocyte-specific genes including peroxisome proliferator activated receptor-γ (PPARγ), CCAAT/enhancer binding protein-α (C/EBPα) and adipocyte fatty acid binding protein 4 (FABP4). In contrast, vitisin C and vitisin D significantly promoted adipogenesis and increased intracellular lipid accumulation, while the two oligostilbenes markedly increased the expression of adipocyte marker genes. In the present study, we found that vitisin A, vitisin B and cis-vitisin A inhibit the adipogenesis and adipocytes differentiation by their influence on the expression of PPARγ, which leads to subsequenet downregulation of PPARγ mediated adipocyte-specific gene during adipogenesis.