Objective To investigate the expression of Toll-like receptor 4 (TLR4) on INS-1 cells, and explore the effects of lipopolysaccharide (LPS) on viability and insulin secretion of INS-1 cells and expression of TLR4 on INS-1 cells. Methods The expression of TLR4 mRNA and protein in INS-1 cells with or without treatment with 1 mg/L LPS was detected by RT-PCR and Western blotting. After treatment with different mass concentrations of LPS (0.01 mg/L, 0.1 mg/L, 1 mg/L, 5 mg/L and 10 mg/L), the viability of INS-1 cells was detected by CCK8 method, and the insulin secretion of INS-1 cells was determined by GSIS. Results RT-PCR and Western blotting revealed that the expression of TLR4 mRNA and protein in INS-1 cells was significantly increased after treatment with LPS (P<0.05). The viability of INS-1 cells was inhibited in a dose and time dependent manner after treatment with 0.1 to 10 mg/L LPS (P<0.05). The insulin secretion of INS-1 cells was significantly decreased after treatment with 1 mg/L LPS (P<0.05). Conclusion The viability of INS-1 cells is inhibited, and the insulin secretion function of INS-1 cells is decreased after treatment with LPS of certain concentrations. The expression of TLR4 mRNA and protein in INS-1 cells is increased after treatment with LPS. Pancreatic β cells may be damaged by endotoxin in a direct manner.