To investigate the optimal conditions for establishing insulin-resistant 3T3-L1 adipocytes.Dexamethason (DEX), 3-isobutyl-methylxanthine (IBMX) and different concentrations of insulin (10(-8), 10(-7), and 10(-6) mol·L(-1)) were used to induce 3T3-L1 preadipocytes into mature adipocytes identified by oil red O staining. We established insulin- resistant 3T3-L1 adipocytes cell model (IR-3T3-L1) by exposing the cells to 1µmol·L(-1) DEX, and the changes of glucose concen- tration in the cell culture were determined by glucose oxidase-peroxidase (GOD-POD) assay.Treatment of 3T3-L1 cells with DEX, IBMX and 10(-6) mol·L(-1)) insulin for 9 days resulted in the differentiation of >90% of the cells into mature adipocytes. IR-3T3-L1 cells cultured for 96 h in the culture media containing 1 µmol·L(-1) DEX showed significantly increased glucose consumption (P=0.0003) as compared with the control group at 36 h (P<0.001).3T3-L1 cells can be induced into mature adipocytes by exposure to 1 µmol·L(-1) DEX, 0.5 mmol·L(-1) IBMX and 10(-6) mol·L(-1)) insulin. A 96 h exposure to 1 µmol·L(-1) DEX can induce 3T3-L1 adipocytes to acquire insulin resistance that can be maintained for 36 h.