Objective To explore whether an excessive accumulation of lipids is involved in the suppression of β cell proliferation and to determine the role of glucose in this experiment. Methods The proliferation of the Ins-1E cells was studied by 3H-thymidine incorporation. The accumulation of fatty acids in beta cells was studied by 3H-palmitate incorporation. Using Taqman real-time RT-PCR, we measured the expression of carnitine palmitoyltransferase-1 (CPT-1) gene. Results As compared with 3.3 mmol/L glucose, the 6.6 mmol/L and 11 mmol/L glucose increased the cell proliferation by 2.27 and 3.04 folds (P<0.05), and the supplemented 0.4 mmol/L palmitate blunted the glucose-induced cell proliferation (1.1 and 0.86 fold, at 6.6 and 11 mmol/L glucose, respectively). With increasing glucose levels, the incorporation of palmitate was augmented,which ranged from 15% to 92%. Wy 14 643 and Triacsin C alleviated the palmitate-induced inhibition of cell proliferation and reduced the incorporation of palmitate. Glucose did not change the expression of CPT-1 gene, but inhibited the palmitate-induced expression in a dose dependent manner. Conclusions The FA-stimulated CPT-1 gene expression is inhibited by glucose, which increases the FA accumulation in β cells and inhibits their proliferation. This could be underlying the mechanism of glucose-dependent lipotoxicity.