The 1,25-dihydroxyvitamin D₃ (1,25D) metabolite increases calcium in blood and expression of inducible nitric oxide synthase (iNOS) and β-defensin 7 (DEFB7) genes in milk somatic cells of healthy cows. Immune cells in the mammary gland produce 1,25D from 25-hydroxyvitamin D₃ (25D) without affecting serum calcium; so, we hypothesized 25D treatment would alter gene expression 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 gene expression and serum calcium. Lactating cows received intramammary injections with 100 or 500 mg 25D, or 10 or 50 mg 1,25D (n = 4 cows/dose) in 2 ipsilateral quarters and a placebo in the other 2 ipsilateral quarters after milking. Control cows (n = 4) received placebo treatments in 2 ipsilateral quarters. Milk samples were collected from each quarter at 0, 6, 12, 24, and 48 h relative to the start of treatments. Blood samples were collected at 0, 12, 24, and 48 h. Data were analyzed by ANOVA using mixed models. Significance was declared at P < 0.05. The 50 μg 1,25D treatment, but not other treatments, increased serum calcium compared with control cows. All treatments increased vitamin D 24-hydroxylase (CYP24A1) in milk somatic cells at 6 h in which the 50 mg 1,25D treatment had the greatest (198-fold) increase. CYP24A1 remained elevated in 500 mg 25D quarters at 24 h but did not differ from control for other treatments at 24 h. The 50 mg 1,25D and 500 mg 25D treatments increased iNOS expression at 6 h. Expression of iNOS remained elevated for 500 mg 25D treatment at 24 h but did not differ from control for 50 mg 1,25D treatment at 24 h. The 10 mg 1,25D and 100 mg 25D treatments did not increase iNOS expression compared with control and DEFB7 in treatment quarters did not differ from control. In conclusion, increasing the intramammary dose of 1,25D from 10 mg to 50 mg increased serum calcium. Intramammary 1,25D increased expression of CYP24A1 and iNOS genes in milk somatic cells to a greater extent than 25D, but duration of increase was greatest for the 500 mg 25D treatment.