Background: In anaesthetised horses, bradycardia secondary to high vagal tone can reduce cardiac output and blood pressure. The use of anticholinergics in horses is limited due to concerns about ileus and abdominal discomfort. This retrospective study sought to determine the prevalence of post-operative abdominal discomfort in healthy horses that received atropine under isoflurane anaesthesia., Study Design: A retrospective evaluation of 222 general anaesthesia events between January 2019 and December 2019 was undertaken., Methods: One hundred and eleven horses that received atropine were identified, and 111 case match controls that did not receive atropine were also selected. Information gathered from the medical records included signalment, anaesthetic drugs, surgical procedures, duration of anaesthesia and surgery, dobutamine and atropine administration, and the occurrence of abdominal discomfort for 24 h after anaesthesia. After initial data analysis, a second cohort of records was assessed separately. The horses in this group were castrated under general anaesthesia (with or without atropine; n = 68). Logistic regression models and Fisher's exact tests were used to look for factors contributing to abdominal discomfort post-anaesthesia. The significance level was set to 5% (p < 0.05)., Results: Atropine administration was not associated with the development of post-anaesthetic abdominal discomfort (OR = 2.121, 95% CI [0.767, 5.869]; p = 0.2). Overall, 18/222 (8.1%) incidences of abdominal discomfort were identified. All incidents occurred in colts undergoing castration. In a separate analysis of only horses anaesthetised for castration, atropine was associated with developing abdominal discomfort (OR = 3.143, 95% CI [1.082, 9.132]; p = 0.04)., Discussion: Atropine was not associated with post-operative abdominal discomfort except in colts undergoing castration. All episodes of discomfort were mild and resolved with minimal intervention. The potential impact of insufficient analgesia in horses undergoing castration is a confounding factor and requires prospective investigation., Conclusion: Overall, atropine at a dose of 0.006 mg/kg IV appears to be a safe method to treat bradycardia in otherwise healthy horses anaesthetised for orthopaedic and upper airway procedures. Further work is required to determine if atropine is safe for colts undergoing castration., (© 2024 The Author(s). Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)