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MicroRNAs (miRNAs) are a class of small endogenous single-stranded noncoding RNA molecules that have important roles in several biological processes. Research in human and laboratory animals has shown that miRNAs can regulate genes associated with type 2 diabetes mellitus and metabolic syndrome, and that the levels of specific miRNAs circulating in the bloodstream can serve as potential biomarkers for the diagnosis and prognosis of these diseases. We hypothesized that insulin-resistant (IR) horses would have a different circulating miRNA profile than those that are healthy. Fifteen nonpregnant mares housed at the Virginia Tech Middelburg Agricultural Research and Extension Center were evaluated for insulin sensitivity, with the frequent sampling intravenous glucose tolerance test. Selected mares, representing the most insulin-sensitive (IS, n = 3) and IR (n = 3) states, and paired for age, weight, and body condition, underwent miRNA profiling. Serum samples were collected, miRNA extracted, and microarray analysis performed to investigate the presence and relative amount of 340 equine miRNAs. Confirmation by quantitative real-time polymerase chain reaction revealed that miRNA was present in the serum of all animals. Results demonstrated different miRNA profiles between groups: Six miRNAs were expressed only in IS mares, five miRNAs were found to have lower quantity in IR mares relative to the IS ones, and three miRNAs were higher quantity in IR mares relative to the IS ones. The novel results of this preliminary study suggest potential new tools that could be developed for the diagnosis and treatment of metabolic syndrome in horses.The use of microRNA as bio markers for insulin resistance in horses.Comparison of circulating microRNA profile in three insulin-resistant and insulin-sensitive horses characterized by a gold standard test.Evidence of different microRNA profiles in insulin-resistant versus insulin-sensitive horses.MicroRNAs found to have relation with insulin-related pathways.