OBJECTIVE: To explore the effect of high glucose on glucose transport activity, protein kinase B (PKB) activity and glucose transporter 4 (GLUT4) in primary cultured rat adipocytes. METHODS: Isolated rat adipocytes were cultured for 24 h at different glucose concentrations (5, 10, 15 and 25 mmol.L-1). The glucose uptake, cellular and membrane GLUT4 expression, PKB protein expression, and PKB serine phosphorylation and activity were measured. RESULTS: These adipocytes treated with glucose of different concentrations showed that high glucose impaired glucose uptake, PKB phosphorylation and activity, and up-regulated GLUT4 translocation, but didn't affect protein expression of PKB and GLUT4. CONCLUSION: High glucose can induce insulin resistance; the mechanism may be involved in the effect of high glucose on PKB serine phosphorylation and activity as well as GLUT4 function.