The objectives of this experiment were to determine the effects of feeding supplemental 25-hydroxyvitamin D₃ (25D) on concentrations of 25D and minerals in serum, lactation performance, and mastitis resistance in dairy cows. Sixty Holstein cows (multiparous, pregnant, lactating, SCC <165,000 cells/mL) were blocked by milk yield and, within each block, randomly assigned to receive a daily dietary supplement containing 1 mg vitamin D (1mgD), 1 mg 25D (1mg25D), 3 mg vitamin D (3mgD), or 3 mg 25D (3mg25D) for 28 d (n = 15/group). Blood and milk were sampled at 0, 7, 14, and 21 d for measurement of vitamin D metabolites, minerals, and energy metabolites in serum. At 21 d, cows fed 1mgD and 3mg25D received an intramammary Streptococcus uberis challenge. Data were analyzed by ANOVA with mixed models using the MIXED procedure of SAS. Significance was declared at P < 0.05. The 1mg25D and 3mg25D cows had greater serum 25D concentrations at 7, 14 and 21 d, greater serum 24,25-hydroxyvitamin D (24,25D) at 21 d, and lower vitamin D at 21 d compared with cows fed 1mgD and 3mgD (25D = 62 ± 7, 66 ± 8 ng/mL, 135 ± 15, and 232 ± 26 ng/mL; 24,25D = 4.9, 4.0, 11.8, and 30.6 ± 2.8 ng/mL; vitamin D = 7.6, 15.7, 1.9, and 3.1 ± 1.6 ng/mL for 1mgD, 3mgD, 1mg25D and 3mg25D, respectively, at 21d). The 3mg25D cows had greater concentrations of Ca and P at 21 d compared with other treatments, which did not differ (Ca = 2.38, 2.4, 2.37, 2.48 ± 0.02 mM; P = 1.69, 1.87, 1.88 and 2.10 ± 0.08 mM for 1mgD, 3mgD, 1mg25D and 3mg25D, respectively). Milk yield and components, DMI, BW, NEFA, BHBA, glucose, 1,25-dihydroxyvitamin D and Mg did not differ between treatments. The 3mg25D cows had less severe mastitis at 60 and 72 h after challenge with S. uberis compared with 1mgD cows. The 3mg25D cows also had slightly lower (P = 0.06) rectal temperature compared with 1mgD cows during the challenge period (38.9 vs. 39.1°C). Feeding 25D increases serum 25D more effectively than supplemental vitamin D, resulting in increased serum mineral concentrations and less severe mastitis in lactating dairy cows.