Milk enriched with vitamin D by supplementing dairy cow diets could provide a valuable dietary source of vitamin D, but information on the feasibility of this approach is limited. In the current study, the effects of supplementing dairy cows with either vitamin D3 or 25(OH)D3 over the transition/early lactation period on plasma and milk vitamin D concentrations were compared. Sixty dairy cows were randomly allocated to 1 of 4 dietary treatments from 14 d precalving to 56 d postcalving. Treatments were a control diet (control) for both precalving and postcalving periods containing 0.625 mg/d of vitamin D3; a precalving diet supplemented with 6 mg of 25(OH)D3/d, but with a postcalving diet matching that of the control diet [25(OH)D3 precalving]; the control diet precalving but with the postcalving diet supplemented with 2 mg of vitamin D3/d (D3max), and the control diet precalving but with the postcalving diet supplemented with 1.5 mg of 25(OH)D3/d [25(OH)D3 postcalving]. No treatment effect on milk yield, composition or 25(OH)D3 concentration was observed. However, an interaction was observed of treatment and time for plasma 25(OH)D3 concentration; this increased within 2 wk of supplementation for the 25(OH)D3 precalving treatment (peaking just after calving, 202 ng/mL), whereas that of the 25(OH)D3 postcalving group had a slower response following supplementation, continuing to increase at 56 d. Correlations were observed between plasma and milk 25(OH)D3 concentrations at d 4 and 14 of lactation, but not at later sampling times. The D3max treatment did not increase 25(OH)D3 concentration in plasma or milk. Overall, results from this study indicate that supplemental 25(OH)D3 is an effective means of enhancing dairy cow plasma 25(OH)D3 concentrations compared with vitamin D3 supplementation, but not necessarily milk concentrations.