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Magnesium metabolism, especially the intracellular magnesium concentration [Mg2+]i, plays an important role in human diabetes mellitus. The aim of this study was to evaluate [Mg2+]i in horses with insulin resistance and to examine a possible association with other markers of insulin dysregulation. The study included 38 horses with a positive combined glucose insulin tolerance test (CGIT) and phenotypic signs of EMS. Baseline glucose (G0), glucose curve, baseline insulin (I0), insulin after 45 minutes (I45), total serum magnesium concentration and [Mg2+]i were measured. The reciprocal inverse square of insulin (RISQI), the modified insulin-to-glucose ratio (MIRG) and the insulin difference ( I45–0) were calculated. A t-test for independent samples was used to compare [Mg2+]i in healthy and EMS horses. Linear regression analyses with uni- and multivariable models were calculated to evaluate the influence of [Mg2+]i on all other blood and clinical parameters. Compared to healthy controls, [Mg2+]i was significantly lower in horses with EMS (P = 0.015). Univariable linear regression models showed significant associations between I0 (P = 0.004, b = 0.006), I45 (P = 0.006, b = 0.001), I45–0 (P = 0.008, b = 0.001), RISQI (P = 0.009, b = –0.158), MIRG (P = 0.001, b = 0.017) and [Mg2+]i. The multivariable model revealed that MIRG significantly influenced [Mg2+]i whereas the other factors had no additional influence. Horses with EMS exhibited an intracellular magnesium deficit that can be partially reversed by increased secretion of insulin. These findings substantiate the crucial role for insulin in regulating [Mg2+]i and its relevance in EMS.