Isoflupredone acetate (IPA) is a synthetic glucocorticoid widely used in equine medicine for its anti-inflammatory properties. While the pharmacokinetics of IPA following intra-articular administration to horses has been well described, studies characterizing intramuscular (IM) administration are lacking. Hypokalemia has been reported following prolonged IPA administration, however, the electrolyte effects of a single dose have not been characterized. This study describes the pharmacokinetics and evaluate changes in plasma electrolyte concentrations and fractional excretion (FE) following a single IM dose of IPA to horses. We hypothesized that a single IPA dose would alter plasma potassium concentrations and associated FEs. Twelve healthy Thoroughbred horses received a 20 mg IM dose of IPA. Blood and urine samples were collected prior to administration and up to 312 hours post, with urine samples collected for electrolyte determination at 72 hours. Drug concentrations were determined by liquid chromatography–mass spectrometry and non-compartmental analysis was conducted. Plasma and urinary electrolytes were measured using ion-selective electrodes. The Cmax, Tmax, and terminal half-life of isoflupredone were 1.55 ± 0.43 ng/mL, 3.50 hours (0.16-5.0 hours; median and range), and 39.6 ± 22.1 hours, respectively. Mean serum potassium decreased significantly from 3.8 ± 0.67 mmol/L (baseline) to 2.7 ± 0.71 mmol/L (P = 0.0006), while serum sodium increased from 137 (135–139) mmol/L to 140 (138–142) mmol/L (P < 0.0001). Potassium FE rose from 39 (20–61)% to 68.92 (27–73)% (P = 0.03), whereas sodium FE decreased from 2.3 (1.47–3.72)% to 1.61 (0.51–3.03)% (P = 0.03). These findings demonstrate that a single IM administration of IPA in healthy horses induces significant hypokalemia and hypernatremia within 72 hours, consistent with observed changes in FEs. The results suggest that even a single IM dose of IPA can produce clinically relevant electrolyte alterations, warranting consideration in therapeutic decision-making and monitoring protocols.