Little information is available regarding the effect of supplementing 25-hydroxyvitamin D3 during the transition period combined with a postpartum oral calcium bolus on Ca homeostasis. The objectives of the current study were to evaluate the effects of 25-hydroxyvitamin D3 combined with postpartum oral calcium bolus on lactation performance, serum minerals and vitamin D3 metabolites, blood biochemistry, and antioxidant and immune function in multiparous dairy cows. To evaluate the effects of 25-hydroxyvitamin D3 combined with oral calcium, 48 multiparous Holstein cows were randomly assigned to one of four treatments: (1) supplementing 240 mg/day vitamin D3 without a postpartum oral Ca bolus (control), (2) supplementing 240 mg/day vitamin D3 with an oral Ca bolus containing 90 g of Ca immediately post-calving (Ca + VitD), (3) supplementing 6 g/day 25-hydroxyvitamin D3 without an oral Ca bolus (25D), and (4) supplementing 6 g/day 25-hydroxyvitamin D3 with an oral Ca bolus containing 90 g of Ca immediately post-calving (Ca + 25D). Lactation performance during the first 21 days was measured. Blood was collected at the initiation of calving and then 1, 2, 7, 14, and 21 days relative to the calving date. The yield of milk (0.05 < p < 0.10), energy-corrected milk (p < 0.05), 3.5% fat-corrected milk (p < 0.05), and milk protein (p < 0.05) were significantly higher in 25-hydroxyvitamin D3-treated groups within 3 weeks of lactation than in vitamin D3-treated cows. The iCa (p < 0.05) and tCa (p < 0.05) were higher in both Ca and 25D + Ca cows than in the control and 25D groups within 48 h. The concentrations of serum tCa (p < 0.05), tP (p < 0.05), and 25-hydroxyvitamin D3 (p < 0.05) in 25D and 25D + Ca cows were higher than those in control and Ca cows within 21 days postpartum. Feeding 25-hydroxyvitamin D3 also showed a lower concentration of malondialdehyde (p < 0.05), interleukin 6 (p < 0.05), and tumor necrosis factor-alpha (TNF-α) (p < 0.05), as well as a higher concentration of alkaline phosphatase (p < 0.05), total antioxidant capacity (p < 0.05), and immunoglobulin G (p < 0.05) than vitamin D3. Supplementing Ca bolus also showed lower concentrations of alanine transaminase (p < 0.05) and TNF-α (p < 0.05). In conclusion, supplementing 25-hydroxyvitamin D3 during the transition period combined with a postpartum oral calcium bolus improved lactation performance, Ca homeostasis, and antioxidant and immune function of medium-production dairy cows within 21 days postpartum.