The oral supplementation of dairy cows with calcidiol (25-OH-D₃) can have positive effects on Ca metabolism around calving and in bone and energy metabolism during lactation. The objective of this experiment was to evaluate oral calcidiol supplementation of cows in late lactation. Thirty Holstein cows (230 ± 137 DIM, 8 primiparous) were individually fed a standard TMR for 14 d and were randomly allocated within blocks (15) to an oral dose (3 × /d) of 1 mg/d of calcidiol (HyD. Rovimix HyD, DSM Nutritional Products) or placebo for 84 d. The basal diet contained 1033 IU/kg of DM of cholecalciferol and 0.77% Ca and 0.46% P in DM. The average intake of cholecalciferol was 23,800 IU/d or 35.7 IU/kg of BW. Data were analyzed with Proc Mixed of SAS with a repeated measures approach. The HyD treatment increased milk yield (31.7 vs 30.9 kg/d. P < 0.01, SEM 0.49) and had no effect on DMI (23.0 kg/d. P = 0.73). The 4% FCM was increased by HyD (28.8 vs 27.5 kg/d. P = 0.03, SEM 0.66). Fat % tended (P = 0.07) to be increased (+ 0.10%-units) and protein % tended (P = 0.09) to be reduced (- 0.06%-units) by HyD. The HyD reduced milk SCC from 105,000 to 83,500 cells/mL (P = 0.02, SEM 12.7) suggesting positive action on mammary gland immunity. Feed efficiencies, BCS, and BW gain did not differ (P ≥ 0.41). The concentration of calcidiol in plasma was increased by HyD supplementation (117.5 vs 52.6 ng/mL. P < 0.01, SEM 3.28) and the increase was of larger magnitude on d 84 than on d 28 (P < 0.01 for the interaction of treatment and day). None of the cows had plasma calcidiol below 40 ng/mL, suggesting that the NRC (2001) recommendation of cholecalciferol supplementation (~20,000 UI/d) associated with exposure to sunlight during the second milking of the day was sufficient to maintain adequate calcidiol concentration in plasma. The concentrations of ionized Ca (P = 0.01) and total Ca (P = 0.03) in serum were increased by HyD, but there were significant interactions (P < 0.05) between treatment and day of measurement. The increase in Ca concentrations induced by HyD was pronounced on d 56 and was not detected on d 84. The supplementation of late lactation dairy cows with HyD increased plasma calcidiol and milk yield and reduced milk SCC, at similar DMI.