Objective·To investigate the effects of maternal vitamin D deficiency and supplementation on the immunomodulatory function of Th1/Th2 in offspring rats. Methods·Forty-eight female SD (8-week-old) rats were randomly assigned to three groups, i.e control group (C group, n=16), vitamin D deficiency group (VDD group, n=16), and vitamin D supplementation group (VDS group, n=16). C group was fed a normal diet and VDD and VDS groups were fed a vitamin D deficiency diet. After four weeks, females and males were housed in a 2∶1 ratio. After gestation, C group and VDS group were fed a normal diet and VDD group was still fed a vitamin D deficiency diet. The vitamin D level in female rats was measured using LC-MS/MS at gestational day 10 (second trimester). The vitamin D level, the ratio of Th1/Th2, and expressions of serum IFN-γ and IL-4 in offspring were measured using LC-MS/MS, flow cytometry, and ELISA 4 weeks after birth, respectively. Results·For C group, VDD group, and VDS group, the 25(OH)D levels in female rats were (23.23±2.08), (5.52±2.36), and (11.35±1.84) ng/mL, respectively, while the 25(OH)D levels in offspring were (7.15±1.30), (0.93±0.22), and (6.82±1.37) ng/mL, respectively. The 25(OH)D levels in female rats and their offspring of VDD group were significantly lower than those of C group and VDS group (P<0.05). The Th1/Th2 ratios in offspring of C group, VDD group, and VDS group were 0.90±0.35, 0.24±0.08, and 0.94±0.26, respectively. The Th1/Th2 ratio in VDD group was significantly polarized toward Th2 and polarization of Th2 in VDS group was remarkably improved compared with C group. The IFN-γ levels in C group, VDD group, and VDS group were (18.72±5.66), (8.27±3.86), and (19.31±5.68) pg/ml, and the IL-4 levels were (3.49±0.52), (4.14±0.55), and (3.52±0.52) pg/ml. The IFN-γ level significantly decreased and the IL-4 level significantly increased in VDD group compared with C group and VDS group (P<0.05). Conclusion·Vitamin D deficiency in pregnant rats caused the imbalance of Th1/Th2 (Th2 polarization) and IFN-γ/IL-4 (drifting toward IL-4) in offspring. Vitamin D supplementation significantly improved the imbalance of Th1/Th2 and IFN-γ/IL-4 in offspring.