The objective of the present study was to investigate the expression levels of toll-like receptor 4 (TLR4) in glioma cells and the mechanisms underlying its regulatory effects on proliferation, migration and apoptosis of glioma cells. A total of three TLR4 silencing short hairpin (sh)RNA plasmids were established, and Lipofectamine® was used to the transfect the human glioma cell line U-87MG. Transfection efficiency was measured via flow cytometry. The interference plasmid exhibiting the largest silencing effect on TLR4 was screened for subsequent experiments using puromycin. Reverse transcription-quantitative PCR and western blot analysis were used to detect the TLR4 gene and protein expression levels, respectively, in stably transfected cells. Flow cytometry measured cell cycle and apoptosis and a wound healing assay was employed to assess the migration ability of transfected cells. The proliferation of transfected cells was detected using Cell Counting Kit-8 assay. TLR4-sh2 exhibited the highest transfection efficiency. Following transfection of U-87MG cells with TLR4-sh2 and negative control (NC) plasmids for 48 h and screening by puromycin, stable transfected cells were named U-87MG-Sh and U-87MG-NC cells respectively. The TLR4 gene and protein expression levels in the U-87MG-Sh cells were significantly lower than in U-87MG and U-87MG-NC cells. The apoptosis rate and the percentage of G0/1 cells were significantly higher, whereas the cell proliferation rate was notably lower, in U-87MG-Sh cells than in the U-87MG-NC and U-87MG cells. The proliferation rate and the cell migration ability of U-87MG-Sh cells were significantly lower than those of U-87MG-NC and U-87MG cells. TLR4 is associated with the proliferation of glioma cells. Inhibition of TLR4 expression levels significantly inhibited proliferation of glioma cells and induced apoptosis. The present study provided insights into the mechanisms associated with the development, progression and invasion ability of glioma cells. Copyright: © Liu et al.