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We investigated the impact of cholecalciferol treatment on Ca homeostasis, vitamin D metabolism, uterine health and milk production of multiparous Holstein dairy cows randomly assigned to a control group (CON; n = 52) or one of 2 treatment groups receiving either 12 × 106 IU (12VitD; recommended by the manufacturer; n = 52) or 6 × 106 IU cholecalciferol (6VitD; n = 54) intramuscularly on d 275 of gestation. Based on former results, we hypothesized that 12VitD cows would show an improved Ca homeostasis but impaired milk production and that half of the recommended dose (6VitD) improves Ca homeostasis without adverse effects. Ca concentrations were determined on d 1, 2, 3 and 5 after calving. For a subsample of 30 cows entering the 3rd lactation (n = 10 per group), samples were analyzed for cholecalciferol (D3), 25-hydroxycholecalciferol (25OHD3), 1,25- and 24,25-dihydroxycholecalciferol (1,25OH2D3 and 24,25OH2D3). Repeated-measures ANOVA was performed to evaluate the treatment effect on blood metabolites and milk yield. Binary outcomes were analyzed using a Chi2 test. Compared with CON, 12VitD cows had increased Ca concentrations on d 1 (P < 0.001), 2 (P = 0.012), and 3 (P = 0.022), and 6VitD cows on d 1 (P = 0.002). At all sampling time points, D3, 25OHD3, and 24,25OH2D3 concentrations of 12VitD and 6VitD cows were higher, compared with CON cows (P-values < 0.001). Levels of 24,25OH2D3 increased linearly with 25OHD3. In respect to 1,25OH2D3, an increase until d 3 was observed in all cows. Compared with CON, 12VitD cows had lower concentrations on d 3 (P = 0.010) and d 5 (P = 0.014). In contrast, 6VitD cows only had lower concentrations on d 5 (P = 0.042). Incidences for retained placenta (1.9 vs. 29.6%; P < 0.001) and metritis (11.5 vs. 31.5%; P = 0.016) were different between CON and 12VitD cows. Milk production of 12VitD cows was lower than milk production of CON cows on the 1st (P = 0.043) and 3rd (P = 0.001) test-day. Our data indicate that treatment with 12VitD can have detrimental effects on uterine health and milk yield, although Ca homeostasis was improved.