Around parturition, many dairy cows experience varying degrees of hypocalcemia, which increases the incidence of several diseases in early lactation. In the current study, an established concept of feeding a diet negative in cation-anion difference (DCAD) was combined with oral supplementation of 25-hydroxyvitamin D(3) (25-OHD(3)) from d 270 of gestation until parturition. Fifty-six dairy cows were divided into 2 feeding groups (low DCAD and control). Fourteen animals of each group received a daily dosage of 3mg of 25-OHD(3). From the beginning of the treatment to d 10 after parturition, plasma samples for analysis of 25-OHD(3), 1,25-dihydroxyvitamin D(3), parathyroid hormone (PTH), Ca(2+), phosphate, the bone resorption marker CrossLaps, and osteocalcin were collected every other day, at calving, and at 6, 12, and 24h after calving. Urine samples for determination of macrominerals and measures of acid-base status were collected on d 6 of treatment and on d 6 after calving. The induction of a compensated metabolic acidosis by the animals on the DCAD diet could be demonstrated by decreased urinary pH. A linear correlation between treatment duration and the plasma concentration of 25-OHD(3) indicated effective absorption of 25-OHD(3) in supplemented animals. The mean plasma concentrations of Ca(2+) from d -4 prepartum to d 4 postpartum were significantly higher in animals treated with the combination of the low DCAD diet and 25-OHD(3) supplementation (1.24±0.02 mmol/mL) compared with the 3 other groups (low DCAD: 1.17±0.02 mmol/mL; control diet plus 25-OHD(3): 1.16±0.02 mmol/mL; control diet: 1.18±0.02 mmol/mL). We postulate that the increased tissue responsiveness to parathyroid hormone induced by the low DCAD is crucial for the observed positive effects of the 25-OHD(3) treatment.