Ridden horses have been reported to be fearful of cows. We tested whether cows could provoke behavioural and cardiac fear responses in horses, and whether these responses differ in magnitude to those shown to other potential dangers. Twenty horses were exposed to cow, a mobile object or no object. The time spent at different distances from the stimulus was measured. In a separate test, heart rate (HR), root mean square of successive differences between heartbeats (RMSSD) and the horses’ perceived fear were assessed at various distances from the stimuli. The horses avoided the area nearest to all stimuli. During hand-leading, the cow elicited the highest HR and lowest RMSSD. Led horses’ responses to the cow and box were rated as more fearful as the distance to the stimulus decreased. Mares had a higher HR than geldings across all tests. HR positively correlated with the fearfulness rating at the furthest distance from the cow and box, and RMSSD negatively correlated with this rating in cow and control conditions. Our results show that these horses’ avoidance response to cows was similar or higher to that shown towards a novel moving object, demonstrating that potentially, both neophobia and heterospecific communication play a role in this reaction.