Objective The aim of this study was to survey the influence of Toll-like receptor 2(TLR2) and Tolllike receptor 4(TLR4) repression to receptor activator of nuclear factor-κB ligand(RANKL) expression of human periodontal ligament fibroblasts(HPDLFs) under the stimulation of lipopolysaccharide(LPS). Methods The level of RANKL in HPDLFs stimulated by 100 ng·mL-1, 1 μg·mL-1 and 10 μg·mL-1 Escherichia coli(E. coli) LPS after 6, 12, 24 and 48 h was detected by enzyme linked immunosorbent assay(ELISA). The level of RANKL in HPDLFs stimulated by 1 μg·mL-1 E. coli LPS after pretreatment with different titre anti-TLR2+anti-TLR4, anti-TLR2 and anti-TLR4 antibody were observed respectively. Results RANKL was detected at 6 h after stimulation with LPS, and the levels of these cytokine were highest at 24 h, and then gradually decreased. The regularity of each LPS concentration was approximately similar. After pretreatment with anti-TLR2+anti-TLR4, anti-TLR2 and anti-TLR4 antibody, the level of RANKL was significantly decreased under the stimulation of 1 μg·mL-1 LPS(Pand TLR4 participate in the process of RANKL expression in HPDLFs induced by LPS. Anti-TLR4 antibody has better inhibition effect to RANKL expression of HPDLFs stimulated by LPS than anti-TLR2.