Background: Flumazenil antagonises the actions of benzodiazepines. There has been no prior research specifically investigating this anaesthetic reversal agent for horses. Objectives: To determine the effects of flumazenil administration in horses on (a) ventilatory parameters after midazolam-ketamine induction and maintenance with isoflurane in oxygen and on (b) the characteristics of recovery from general anaesthesia. Study design: Blinded, randomised, crossover experiment. Methods: Six horses were randomly assigned to receive high-dose flumazenil (F_high, 20 µg/kg), low-dose flumazenil (F_low, 10 µg/kg) and saline (control). Cardioventilatory parameters were monitored. After 90 minutes of isoflurane anaesthesia, a bolus of F_high, F_low or saline was administered i.v. The horses were recovered using head and tail rope assistance. The times to first movement, to achievement of sternal recumbency, to the first attempt to stand and the total recovery time were determined. The recovery quality was evaluated using a 115-point recovery scoring system. The cardioventilatory parameters and recovery times were analysed using mixed-effects regression analyses. Intraclass correlation (ICC) analysis was used to evaluate the recovery scores. A Mann-Whitney U test assessed the relationship between recovery score and flumazenil administration. Results: A significant difference with flumazenil administration was found for SpO₂, mean arterial pressure, I:E ratio, minute volume of ventilation (MV) and peak inspiratory pressure. There was a significant difference with flumazenil administration for the time to sternal recumbency, the time to the first attempt to rise and the total recovery time. There was no significant difference in total recovery score with flumazenil administration. Main limitations: Plasma levels of midazolam and flumazenil were not obtained. Conclusions: Flumazenil has a dose-dependent effect on MV and recovery time, which may make it useful in cases for which a prolonged anaesthetic recovery is undesirable.