In humans, facial expressions are rich sources of social information and have an important role in regulating social interactions. However, the extent to which this is true in non-human animals, and particularly in non-primates, remains largely unknown. Therefore we tested whether domestic horses (Equus caballus) could discriminate between facial expressions of their conspecifics captured in different contexts, and whether viewing these expressions elicited functionally relevant reactions. Horses were more likely to approach photographic stimuli displaying facial expressions associated with positive attention and relaxation, and to avoid stimuli displaying an expression associated with aggression. Moreover, differing patterns of heart rate changes were observed in response to viewing the positive anticipation and agonistic facial expressions. These results indicate that horses spontaneously discriminate between photographs of unknown conspecifics portraying different facial expressions, showing appropriate behavioural and physiological responses. Thus horses, an animal far-removed from the primate lineage, also have the ability to use facial expressions as a means of gaining social information and potentially regulating social interactions.