Objective: To observe the glucose transportation and Glut4 expression in the induction of insulin resistance in 3T3-L1 adipocytes by three methods (dexamethasone, free fatty acids, and high glucose/hyperinsulinism). Methods: Insulin resistance was induced in three different model groups of 3T3-L1 adipocytes according to the three different methods. The glucose transportation rate was detected by the uptake of 2-deoxy-[ 3h]-D-glucose in each group after 12, 24, 36, 48 and 60 hours. The Glut4 expression was assayed by means of Western blot after 60 hours. Results: Within 24 hours, as compared with normal control group, the glucose transportation in each model group decreased significantly (P < 0.01). After 24 hours, as compared with that in normal control group, the glucose transportation rate in free fatty acid group showed no difference (P > 0.05), but decreased obviously in dexamethasone group (P < 0.05), while the rate in high glucose and hyperinsulinism group decreased significantly even than that in dexamethasone group (P < 0.01). The Glut4 expression in dexamethasone group and free fatty acids group both decreased significantly as compared with normal control group (P < 0.01). And compared with that respectively in dexamethasone group and free fatty acids group, the Glut4 expression in high glucose and hyperinsulinism group decreased significantly (P < 0.01). Conclusion: High glucose and hyperinsulinism can induce 3T3-L1 adipocytes to produce stronger insulin resistance, and the production of the resistance may be due to the inhibition of Glut4 expression.