OBJECTIVE: To investigate the effect of vitamin D3 on polarization of monocyte macrophages induced by serum from patients with ankylosing spondylitis (AS). METHODS: Twenty AS naïve patients and 20 healthy controls from Wenzhou People's Hospital during January 2016 and December 2017 were enrolled. The macrophages were differentiated from THP1 cells induced by phorbol 12-myristate 13-acetate (PMA), and then co-cultured with the serum from healthy subjects (control group) or AS patients. Vitamin D3 was added in the medium mixed with serum from AS patients. Flow cytometry was used to analyze the ratio of CD68 and CD206 positive cells, and RT-PCR was performed to detect the mRNA expression of inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1). RESULTS: THP1 cells could be polarized into mononuclear-macrophages with the induction of PMA. The proportion of CD206 positive cells in AS-serum group was lower than that in the control group (t=9.434, P<0.05), while the proportion of CD68 positive cells was higher than that in the control group (t=43.920, P<0.05). The proportion of CD206 positive cells in vitamin D3 group was higher than that in AS-serum group (t=8.895, P<0.05), while the proportion of CD68 positive cells was lower than that in AS-serum group (t=9.089, P<0.05). mRNA expression of Arg-1 in AS-serum group was lower than that in the control group (t=8.899, P<0.05), while mRNA expression of iNOS was higher than that in the control group (t=3.656, P<0.05). mRNA expression of Arg-1 in vitamin D3 group was higher than that in AS-serum group (t=6.219, P<0.05), while mRNA expression of iNOS was lower than that in AS-serum group (t=5.876, P<0.05). CONCLUSIONS: Vitamin D3 can regulate the polarization of mononuclear macrophages for immunoregulation in patients with AS.