ABSTRACTAim: To determine if vitamin D3 treatment reduced the incidence of vaginal prolapse in pregnant sheep on a North Canterbury sheep breeding property.Methods: Pregnant ewes from a single farm were allocated to three treatment groups in May 2018. At this time, the first group (EarlyVitADE; n = 512) received an I/M 1 mL dose of 500,000 IU/mL vitamin D3, 60,000 IU/mL vitamin A, and 25 mg/mL vitamin E. This was repeated in July 2018, when the second group (LateVitADE; n = 695) also received the same treatment. The third group (n = 737) were untreated controls. All cases of vaginal prolapse on the property were recorded from pregnancy diagnosis in June 2018 until ewes were set-stocked in August 2018. The planned start of lambing was 10 August 2018.Results: During the period of observation, vaginal prolapses were recorded in 3/699 (0.4%) 2-year-old ewes, and the odds of vaginal prolapse were not associated with treatment group in these ewes (p > 0.3). Amongst ewes aged ≥3 years, during the same period, there were 6/333 (1.8%), 6/443 (1.4%) and 25/469 (5.3%) cases in the EarlyVitADE, LateVitADE and control groups, respectively. Compared to control ewes, the odds of vaginal prolapse were reduced in both the EarlyVitADE (OR = 0.37; 95% CI = 0.15–0.92) and LateVitADE (OR = 0.25; 95% CI = 0.10–0.62) treatment groups.Conclusions and clinical relevance: In this preliminary study, administration of injectable vitamins A, D3, and E to pregnant ewes reduced the incidence of vaginal prolapse during the period from pregnancy diagnosis to set-stocking on one North Canterbury hill-country farm. Due to the restricted data collection period, this investigation should be replicated to better quantify the repeatability of the observed treatment effect over the complete lambing period.