Intramammary 25-hydroxyvitamin D3 (25D) and 1,25-dihydroxyvitamin D3 (1,25D) treatments stimulate immune defenses of the mammary gland. We hypothesized 25D treatment, in contrast to 1,25D, would exert activity in the mammary gland without affecting serum calcium. The objective was to determine the effect of dose and source of intramammary vitamin D treatments on milk somatic cell gene expression and serum calcium. Twenty lactating Holstein cows with somatic cell count <200,000 cells/mL of milk were used for the experiment. Cows were blocked by somatic cell count and randomly assigned to 1 of 5 intramammary treatments (n = 4 cows/treatment): placebo control (CNTRL; 0.4% Tween 20 in phosphate-buffered saline), 100 µg of 25D, 500 µg of 25D, 10 µg of 1,25D, or 50 µg of 1,25D. Treatments were administered in 2 ipsilateral quarters after milking. Blood samples were collected at 0, 12, 24, and 48 h for measurement of Ca and 1,25D. Milk samples were collected from each quarter at 0, 6, 12, 24, and 48 h relative to the start of treatments for measurement of gene expression in milk somatic cells. The 1,25D treatments increased serum concentrations of 1,25D and Ca in a dose-dependent manner with maximum 1,25D and Ca concentrations of 199 ± 6 pg/mL and 2.73 ± 0.04 mM, respectively, observed for 50 µg of 1,25D cows compared with 59 ± 6 pg/mL and 2.54 mM, respectively, for CNTRL cows. The 25D treatments did not affect serum 1,25D and Ca compared with CNTRL. The 25D and 1,25D treatments increased mRNA transcripts for vitamin D 24-hydroxylase (CYP24A1), inducible nitric oxide synthase (NOS2A), and chemokine C-C motif ligand 5 (CCL5) in a dose-dependent manner. The 50 µg of 1,25D treatment resulted in the greatest CYP24A1 expression (303-fold relative to CNTRL) at 6 h but was not different from CNTRL at 24 h. In contrast, CYP24A1 was 57-fold greater for cows that received 500 µg of 25D compared with CNTRL at 24 h. In conclusion, intramammary 25D treatment is effective at regulating gene expression in the mammary gland without systemic effects on serum 1,25D and Ca that occur with intramammary 1,25D treatment.