Uterine tube obstruction is the leading cause of unexplained subfertility in mares. Over the years, numerous techniques were developed to unblock uterine tubes with variable success. In recent years, the administration of misoprostol, a synthetic analog of prostaglandin E1, in the uterus, has regained popularity in equine practice. However, there have been some concerns regarding an exacerbated post-infusion uterine inflammatory response to misoprostol; nonetheless, this has yet to be critically studied. In addition, debate exists when mares should receive misoprostol. This study aimed to determine the uterine inflammatory response in mares after infusion with misoprostol during estrus or diestrus. We hypothesized that misoprostol infusion induces a greater inflammatory response than sham infusion; similarly, diestrus results in a greater inflammatory response than estrus. Forty-four estrous cycles of light breed mares (n=11) were sequentially randomly assigned to receive misoprostol or sham infusion during estrus and diestrus. The misoprostol and sham infusions were performed by directing a flexible pipette (Minitube, Germany) deep into each uterine horns, which received 200mcg of misoprostol diluted in 3 mL of Lactate Ringer's Solution (LRS), whereas the sham inoculation consisted of 3 mL of LRS. The cycles assigned for estrus treatment received it when preovulatory follicles ranged from 28-32 mm, and diestrus cycles were treated eight days post-ovulation. The day after each infusion, mares had uterine lavage performedwith LRS. Uterine edema and intrauterine fluid accumulation were performed by transrectal ultrasonography immediately before (0h), 24, 48, and 72 h after treatments. At similar time points, uterine cytology samples were harvested to count the number of neutrophils in a high-power field (400 ×). Ovulation was hastened with deslorelin (1mg, i.m.) during estrus or the subsequent estrus after diestrus infusion. Mares received 5 mg of dinoprost (i.m.) 24 h after infusion in estrus and diestrus. Statistical analyses were performed with Graph Prisma. Neutrophil counts were analyzed with ANOVA repeated measures and Tukey's as post-hoc. Edema scores and intrauterine fluid accumulation were analyzed with Kruskal-Wallis and Dunn's post-hoc. Embryo recovery rates were evaluated with Fisher's test. There were effects of time (P=0.04) but no effects of group (P=0.96), or stage of the estrous cycle (P=0.26), or interaction between group and time for edema scores (P=0.97). Similarly, there were effects of time (P=0.03) but no effects of group (P=0.32), stage of the estrous cycle (P=0.86), or interactions between group and time or stage of the estrous cycle for neutrophil counts (P=0.87). Uterine fluid scores did not change over time (P=0.39), groups (P=0.22), or stage of the estrous cycle (P=0.77). Embryo recovery was similar for estrus (Misoprostol 46% vs. sham 46%) vs. diestrus (Misoprostol 67% vs. sham 46%) (P>0.05). In conclusion, 200 mcg of misoprostol bilateral administration did not exacerbate uterine inflammation in mares.