Persistent breeding-induced endometritis (PBIE) is one of the leading causes of subfertility in mares, affecting 10-15% of the population. The objective of this study is to identify a novel, antibiotic-free treatment to improve inflammation resolution in mares susceptible to PBIE. Mares susceptible to PBIE tend to be older animals with an impaired immune response, factors that are associated with vitamin D3 (D3) deficiency in humans. As our previous research suggests that vitamin D is actively transported to the site of inflammation where it plays a central role in resolving inflammation, we hypothesized that local administration of vitamin D3 would aid in improved uterine clearance and enhance inflammation resolution in mares susceptible to PBIE. To this end, susceptible (n = 6) and resistant (n = 4) mares were bred with centrifuged, extended frozen-killed semen containing either D3 or vehicle (Con) in a randomized cross-over design, with a rest cycle between. Status was assigned based on endometrial biopsy score (modified Kenney-Doig) and results of a test breeding cycle at the beginning of the season, with susceptible mares having a 2B or 3 biopsy score coupled with 96h+ of fluid retention, while resistant mares had a 1 or 2a biopsy score and cleared fluidby 48 h. Plasma was obtained at the time of insemination (0h), 6h post-breeding (6h) and 7d post-breeding (7d), with endometrial biopsies taken at both 6h and 7d. Susceptible mares showed a nearly 65% decrease (P < 0.001) in the days to uterine clearance when treated with D3 (2.2 ± 0.48) compared to their own Con cycle (6.0 ± 3.1), bringing days to uterine clearance to a level which was not significantly different than the control cycle for resistant mares. Enhanced inflammation resolution was also evident on a histological level, with mucosal inflammation and leukocyte infiltration significantly higher in the control cycle, as compared to the D3 cycle and the pre-breeding biopsy (P <0.05). Lastly, plasma concentrations of IL-1α, IL-6 and IL-10 were altered, with pro-inflammatory IL-1α significantly decreased in D3-treated mares, while anti-inflammatory IL-6 and IL-10 showed a significant increase, with the highest measured levels seen at 7d post-breeding (P<0.05). In conclusion, vitamin D3 shows promising results as a potential treatment for PBIE in mares, showing greatly improved resolution of breeding-induced inflammation. Work is currently underway to determine whether pregnancy rates are influenced by D3 treatment, as well as mechanistic pathways.