Background: To investigate the mechanism of vitamin D level on the regulation of peripheral blood lymphocyte subsets and serum Th1/Th2 cytokines in patients with unexplained recurrent spontaneous abortion (URSA). Methods: Eighty female patients with URSA attending Sichuan Jinxin Xinan Women's and Children's Hospital from January 2020 to May 2021 were selected as the study group, and 30 agematched women with a history of healthy deliveries were chosen as the control group, and peripheral blood lymphocyte subpopulations and serum Th1/Th2 cytokines of people with different levels of vitamin D were detected in the study group by flow cytometry, respectively. The results of immune factors before and after supplementation were analyzed in 40 of these patients with low vitamin D levels. The results of lymphoid subpopulations and Th1/Th2 cytokines in 19 patients with normal pregnancy before and after vitamin D supplementation and after normal pregnancy were also analyzed comparatively. Results: (1) Serum 25(OH)D in the study group was lower than in the control group; peripheral blood Th cells, B cells and NK cells in the study group were higher than in the control group; IL -2, TNF-alpha, IFN-gamma and IL -6 in the study group were higher than in the control group, while IL -4 and IL10 in the study group were lower than in the control group (P < 0.05). (2) Th cells, B cells and NK cells of URSA patients in the vitamin D low level group were higher than those in the vitamin D normal group; serum cytokines IL -2, TNF-alpha and IFN-gamma of patients in the vitamin D low level group were higher than those in the vitamin D normal group (P < 0.05); (3) Th cells, B cells and NK cells in URSA patients after vitamin D supplementation were lower than before vitamin D supplementation; serum cytokines IL -2, TNF-alpha and IFN-gamma after vitamin D supplementation were lower than before vitamin D supplementation, IL -4 and IL -10 after vitamin D supplementation were higher than before vitamin D supplementation (P < 0.05), and there was no significant difference in IL -6 before and after vitamin D supplementation. (4) Th cells, B cells and NK cells in patients with normal pregnancy after vitamin D supplementation and after pregnancy were lower than those before vitamin D supplementation; serum cytokines IL -2, TNF-alpha and IFN-gamma after vitamin D supplementation and after pregnancy were lower than those before vitamin D supplementation, and serum cytokines IL -4 and IL -10 after vitamin D supplementation and after pregnancy were higher than those before vitamin D supplementation, TNF -alpha, IFN-gamma after pregnancy were lower than after vitamin D supplementation (P < 0.05), IL -6 was not significantly different before and after vitamin D supplementation and after pregnancy. Conclusion: Vitamin D deficiency rate was high in URSA patients. Th,B,NK cells and IL -2, TNF alpha, IFN-gamma, IL -6 cytokines were high, while IL -6 and IL -10 were low in URSA patients. IL -2, TNF-alpha, IFN-gamma cytokines and Th, B, NK cells were increased in vitamin D deficient URSA patients, and Vitamin D deficiency may be an important cause or aggravating factor of immune dysfunction in URSA patients. Vitamin D has an immunomodulatory effect on URSA patients, promoting successful pregnancy by down -regulating peripheral blood Th, B, and NK cells and IL -2, TNF-alpha, and IFN-gamma cytokines, while up -regulating IL -4 and IL -10.