The use of corticosteroid esters, such as betamethasone sodium phosphate/betamethasone acetate (BTM-PA) in performance and racehorses is commonplace. While the pharmacokinetics and pharmacodynamics of betamethasone following intra-articular administration to horses have been well described, studies characterizing intramuscular administration are lacking. The objectives of this study were to describe the pharmacokinetics and effects on cortisol suppression following intramuscular administration to horses. Twenty-four exercised Thoroughbred research horses received a single intramuscular dose of 12 mg BTM-PA (BetaVet®). Blood and urine samples were collected at various times post administration for up to 408 hours. Concentrations of betamethasone were determined using liquid chromatography-tandem mass spectrometry and pharmacokinetic parameters determined using a NLME Population PK model. The possible effects of covariates (age, weight, and gender) on between subject variability for the structural pharmacokinetic parameters was assessed. The duration of pharmacodynamic effects was assessed by measuring changes in cortisol. Intramuscular betamethasone administration resulted in low but sustained plasma concentrations. The Cmax, Tmax, and terminal half-life of betamethasone were 6.43 ± 1.70 ng/mL, 0.75 (0.5-2.0 hours; median and range), and 30.5 ± 20.4 hours, respectively. For compartmental modeling, a 3-cmpt model best fit the data and covariates were not found to have significant effects on the variability of pharmacokinetic parameters. Based on Monte Carlo simulations, for a simulated population of 1000 horses, a detection time of 23 days is recommended for betamethasone concentrations to fall below the Racing Medication and Testing Consortium’s currently recommended 10 pg/mL regulatory screening limit, in 99% of the population. Betamethasone urine concentrations were above the limit of quantitation (0.05 ng/mL) in 2/24 horses at 408 hours post administration. Significant suppression of endogenous cortisol was observed for 360 hours post administration. The prolonged detection time warrants an extended withdrawal time for intramuscular administration prior to competition in performance horses.