Objective：To investigate the effect of serum 1,25 dihydroxyvitamin D3 on macrophage polarization in patients with ankylosing spondylitis and its regulation mechanism on Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signal pathway. Methods: 20 newly diagnosed AS patients and 20 healthy subjects who came to the Department of Rheumatology and Immunology of our hospital from March 2018 to March 2019 were selected as the research objects. The human monocyte line THP1 is induced to differentiate into macrophages by phorbol ester. The cell experiment was divided into three groups: normal group, AS group and experimental group. Normal group: THP1 mononuclear macrophage medium is added with 5% serum of control subjects. AS group: THP1 mononuclear macrophage medium was added with 5% serum of AS patients. Experimental group: THP1 mononuclear macrophages were added with 5% serum of AS patients as well as 1 mL of 100nmol/L 1,25-dihydroxyvitamin D3 solution. Flow cytometry was used to detect the changes in the ratio of CD206 and CD68 cells in each group of cells; ELISA method was used to detect the content of interleukin-10(IL-10), IL-6 and tumor necrosis factor-α (TNF-α) in the supernatant of each group of cells; Gene biochip analysis and real-time fluorescence quantitative polymerization Enzyme chain reaction (real-time quantitative polymerase chain reaction, RT-qPCR) analysis were used to determin the target genes of 1,25-dihydroxyvitamin D3; Western blot was used to detect the expression of proteins related to cell signaling pathways in each group. Results: Compared with the normal group, the proportion of CD206-positive cells in the AS group and the experimental group was significantly reduced, the content of IL-10 in the cell supernatant was significantly decreased, the proportion of CD68-positive cells, and the content of IL-6, TNF-α in the cell supernatant were increased significantly, and the differences were statistically significant (all P＜0.05). Compared with the AS group, the proportion of CD206-positive cells in the experimental group and the IL-10 content in the cell supernatant were increased significantly, and the ratio of the CD68-positive cells serotonin and the content of IL-6 and TNF-α in the cell supernatant were significantly reduced, and the difference were statistically significant (all P＜0.05). Gene biochip analysis and RT-qPCR confirmed that TLR4 gene is the target gene of 1,25-dihydroxyvitamin D3. Compared with the normal group, the expressions of TLR4, MyD88, and NF-κB in the cells of the AS group and the experimental group were significantly increased. Compared with the AS group, the expressions of TLR4, MyD88, and NF-κB in the cells of the experimental group were significantly reduced. It is statistically significant (all P＜0.05).Conclusion: 1,25-dihydroxyvitamin D3 can regulate TLR4/MyD88/NF-κB signaling to regulate the polarization process of macrophages in patients with ankylosing spondylitis, and play an immunomodulatory effect.