Myeloperoxidase (MPO), as a marker of neutrophil activation, has been associated with equine endometritis. However, in absence of inflammation, MPO is constantly detected in the uterine lumen of estrous mares. The aim of this study was to characterize MPO in the uterus of mares under physiological conditions as a first step to better understand the role of this enzyme in equine reproduction. Total and active MPO concentrations were determined, by ELISA and SIEFED assay, respectively, in low-volume lavages from mares in estrus (n = 26), diestrus (n = 18) and anestrus (n = 8) in absence of endometritis. Immunohistochemical analysis was performed on 21 endometrial biopsies randomly selected: estrus (n = 11), diestrus (n = 6) and anestrus (n = 4). MPO, although mostly enzymatically inactive, was present in highly variable concentrations in uterine lavages in all studied phases, with elevated concentrations in estrus and anestrus, while in diestrus, concentrations were much lower. Intracytoplasmic immunoexpression of MPO was detected in the endometrial epithelial cells, neutrophils and glandular secretions. Maximal expression was observed during estrus in mid and basal glands with a predominant intracytoplasmic apical reinforcement. In diestrus, immunopositive glands were sporadic. In anestrus, only the luminal epithelium showed residual MPO immunostaining. These results confirm a constant presence of MPO in the uterine lumen of mares in absence of inflammation, probably as part of the uterine mucosal immune system, and suggest that endometrial cells are a source of uterine MPO under physiological cyclic conditions.