Background: Misoprostol is an E prostanoid (EP) 2, 3 and 4 receptor agonist that is anecdotally used to treat and prevent NSAID-induced GI injury in horses. Misoprostol elicits anti-inflammatory effects in vivo in men and rodents, and inhibits TNFa production in equine leucocytes in vitro. Objective: Define the pharmacokinetic parameters of oral misoprostol in horses, and determine the inhibitory effect of oral misoprostol administration on equine leucocyte TNFa production in an ex vivo inflammation model. Study design: Pharmacokinetic study, ex vivo experimental study. Methods: Six healthy adult horses of mixed breeds were used. In phase one, horses were given 5 lg/kg misoprostol orally, and blood was collected at predetermined times for determination of misoprostol free acid (MFA) by UHPLC-MS/MS. Pharmacokinetic parameters were calculated. In phase two, horses were dosed as in phase one, and blood was collected at T0, 0.5, 1 and 4 h following misoprostol administration for leucocyte isolation. Leucocytes were stimulated with 100 ng/mL LPS, and TNFa mRNA concentrations were determined via quantitative real-time PCR. Results: About 5 lg/kg oral misoprostol produced a rapid time to maximum concentration (T max) of 23.4 AE 2.4 min, with a maximum concentration (C max) of 0.29 AE 0.07 ng/mL and area under the curve (AUC 0˰) of 0.4 AE 0.12 h ng/mL. LPS stimulation of equine leucocytes ex vivo significantly increased TNFa mRNA concentrations, and there was no significant effect of misoprostol even at the T max. Main limitations: Only a single dose was used, and sample size was small. Conclusions: Misoprostol is rapidly absorbed following oral administration in horses, and a single 5 lg/kg dose had no significant inhibitory effect on ex vivo LPS-stimulated TNFa mRNA production in leucocytes. Further studies analysing different dosing strategies, including repeat administration or combination with other anti-inflammatory drugs, are warranted.