Objective:To investigate the effect of elevated Toll-like receptor 3(TLR3) on the growth of INS-1 pancreatic β-cell and possible cell cycle changes during this process. Methods:INS-1 cells were synchronized at the G0 phase by serum deprivation for 12 h. Polyinosinic-polycytidylic acid (PIC) was used to activate TLR3,and the proteasome was inhibited with MG132. Cell viability was assessed by MTT colorimetric assay. Cell cycle distribution was measured by FACS analysis,and cell cycle protein Cyclin D1 was further detected using real-time RT-PCR and Western blot analysis. Results:PIC significantly inhibited the viability of INS-1 cells in a dose-dependent manner (P < 0.05). Furthermore,PIC induced an increase in the proportion of cells at the G1 phase and a corresponding decrease in the number of cells at the S phase(P < 0.05). Additionally,PIC treatment caused a significant reduction in Cyclin D1 levels (P < 0.05),but without a parallel decrease in Cyclin D1 mRNA levels(P > 0.05). Inhibition of the proteasome with MG132 rescued Cyclin D1 protein from degradation. Conclusion:The results suggest that TLR3 activation with PIC directly inhibits pancreatic β-cell proliferation through cell cycle arrest at the G1 phase,possibly by downregulation of Cyclin D1 in a proteasome-dependent manner,which ultimately results in an overall β-cell mass deficiency.