Flupirtine (FLU) after Intravenous (IV) and Per Os (PO) administration in healthy horses was evaluated. Six mixed breed adult mares were randomly assigned to two groups using cross-over design (2 x 2 Latin-square). Group 1 received a single dose of 1 mg/kg of FLU injected IV into the jugular vein. Group 2 received FLU (5 mg/kg) via nasogastric tube. The wash out period was 1-week. Blood samples (5 mL) were collected at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 24, 36 and 48 h and plasma was analysed by a validated HPLC method. Some mild and transient adverse effects (that spontaneously resolved within 5 minutes) were observed in 2 out of 6 animals after IV administration. No adverse effects were noticed in the PO administration group. After IV and PO administrations, FLU was detectable in plasma for up to 36 h. The mean elimination half-life was longer after PO (10.27 h) than after IV (3.02 h) administration. The oral bioavailability was about 70%. After in silico pharmacokinetic simulation/modelling, an oral dose of 2.6 mg/kg in horses has been calculated to give C_max and AUC values similar to those reported in humans after a clinical dose administration with a theoretical FLU effective plasma concentration of 187 ng/ml. This study could pave the road for the use of this active ingredient in equine medicine as analgesic after pharmacodynamic studies.