Calves need vitamin D to support bone growth and immunity. Feeding dairy calves pasteurized whole milk is a common practice, but the vitamin D status of milk-fed calves has not been widely appreciated. The objectives of this study were to determine serum 25-hydroxyvitamin D (25D) concentrations of dairy calves fed pasteurized whole milk and the effects of subcutaneous vitamin D injections on serum 25D concentrations of milk-fed dairy calves. Two experiments were conducted on 2 separate farms and serum 25D concentrations were measured using a 25D ELISA. In the first experiment, 23 Holstein calves received a vitamin AD&E (1200 IU α-tocopherol, 400,000 IU retinyl-palmitate, and 40,000 IU vitamin D₃; n = 11) or saline injection (n = 12) at birth, and were fed pasteurized whole milk and housed indoors. Serum 25D concentrations of the control calves were 8.1 ± 1.0, 11.2 ± 2.6, 13.0 ± 3.2, and 13.3 ± 1.6 ng/mL (mean ± SE) at 0 d, 7 d, 22 d, and 37 d of age, respectively. In contrast, serum 25D concentrations of the vitamin AD&E treated calves were the same as control calves at birth (10.0 ± 1.0 ng/mL), greater at 7 d and 22 d (25.4 ± 2.8 and 24.9 ± 3.4 ng/mL, respectively; P < 0.05), and not different from controls at 37 d (14.3 ± 1.6 ng/mL; treatment x time interaction, P < 0.05). In the second experiment, 13 Holstein bull calves received either 80,000 IU of vitamin D₃ via subcutaneous injection at birth and once weekly for 3 weeks (n = 5) or no injection (control, n = 8), and were fed pasteurized whole milk and housed under shaded structures. Serum 25D concentrations of the non-treated calves were 13.4 ± 3.3, 5.1 ± 3.4, 8.0 ± 4.1, and 8.7 ± 4.9 ng/mL (mean ± SE) at 0 d, 7 d, 14 d, and 21 d of age, respectively. In the vitamin D-treated calves, serum 25D was the same as control calves at birth, but was increased to 25.6 ± 4.4, 38.1 ± 5.2, and 41.2 ± 6.2 ng/mL (mean ± SE) at 7 d, 14 d and 21 d of age, respectively (treatment x time interaction, P < 0.01). In conclusion, milk-fed dairy calves, particularly if housed indoors or under shade, are at risk for vitamin D deficiency (serum 25D < 10 ng/mL) if they do not receive supplemental vitamin D, and continuous vitamin D supplementation is needed to maintain vitamin D status of calves.