The objective of this study was to investigate the effects of leukotriene B4 (LTB4) on the expression of interleukin-32 (IL-32) interferon-γ (IFN-γ) and chemokine monocyte chemoattractant protein (MCP-1) and macrophage inhibitory protein (MIP-1α) in rheumatoid arthritis (RA). The rat model of RA collagen induced-arthritis (CIA) was established. The levels of LTB4, interleukin-32, IFN-γ and chemokines MCP-1 and MIP-1α in CIA rats were detected by ELISA. After the rat synovial cells were isolated and treated with different concentrations of LTB4, the effect of LTB4 the expression of IL-32, IFN-γ and chemokines MCP-1 and MIP-1α mRNA in synovial cells was detected by real-time quantitative PCR, the effect of LTB4 on protein expression was detected by immunoblotting. The effects of different concentrations of LTB4 on the viability and apoptosis of synovial cells were detected by LDH and cell proliferation reagent WST-1. Compared with the control group, the levels of LTB4, IL-32, IFN-γ and chemokines MCP-1 and MIP-1α were significantly increased in the serum of the CIA group. After treatment of CIA rat synovial cells with different concentrations of LTB4, the expression of IL-32, IFN-γ and chemokines MCP-1 and MIP-1α mRNA and protein were increased with significant differences among groups. WST-1 and flow cytometry showed that LTB4 had significant toxic effects on synovial cells and promoted apoptosis. In conclusion, LTB4 promotes the expression of interleukin-32, IFN-γ and chemokines MCP-1 and MIP-1α in synovial cells and facilitates apoptosis of synovial cells.