The objective of this experiment was to determine the effects of dietary vitamin D source on serum calcium (Ca), urinary Ca excretion and milk production when fed in combination with a prepartum acidogenic negative DCAD diet. Non-lactating, pregnant multiparous cows (n=15), balanced for breed (Holstein n = 9 and Jersey n = 6) and previous mature equivalent milk production, were assigned to 1 of 3 treatments (5 cows/treatment), consisting of a control (PCH; positive DCAD, 8.9 mEq/100g DM) and two negative DCAD diets (-15.4 mEq/100g DM), one with vitamin D3 (Cholecalciferol; NCH), and one with 25-hydroxyvitamin D3 (calcidiol; NCA; DSM Nutritional Products). The treatments were formulated to provide 1.95 mg/d of vitamin D and were fed 28 d prior to expected calving date. Delivery of vitamin D sources was accomplished by manufacture of a pellet and 2 kg of these pellets were individually fed simultaneously each day along with 2 kg of ground corn daily at 0800. Negative DCAD treatments were formulated to provide 0.46 kg/d of Animate (Phibro Animal Health) and, if needed, additional Animate was top-dressed at each feeding to achieve a urine pH between 5.5 – 6.0 based on the previous day’s urine pH. Close-up cows had ad libitum access to chopped bermudagrass (Cynodon dactylon L.) hay and hay intake was measured using SmartFeed Pro systems (C-Lock Inc.; Rapid City, SD). Prepartum urine and serum samples were collected weekly and serum was collected 36, 48, and 72 h post-calving. Prepartum DMI as a percent of body weight was not (P = 0.66) affected by treatments. Cows fed NCH and NCA had greater (P = 0.02) prepartum serum Ca than PCH and tended to have greater urinary Ca excretions (P = 0.10). Average postpartum serum Ca (mg/dL) was greater (P = 0.05) for cows fed NCH (8.8) compared with PCH (7.8), whereas NCA (8.4) was numerically intermediate and not (P > 0.05) different from either of the other treatments. Postpartum DMI was not affected by treatment (P = 0.39). Daily MY (kg/d) was greatest (P < 0.01) for NCA (37.5) compared with the other treatments and NCH (34.1) was intermediate and greater than PCH (29.9). These results suggest that an acidogenic prepartum diet in combination with vitamin D was effective in maintaining peripartum serum Ca and the 25-hydroxy form of vitamin D improved MY compared with NCH in early lactation.