α2 agonists are frequently used in horses with colic, even though they have been shown to inhibit gastrointestinal motility. The aim of this study was to determine the effect of dexmedetomidine on small intestinal in vitro contractility during different phases of ischaemia. Experimental segmental jejunal ischaemia was induced in 12 horses under general anaesthesia, and intestinal samples were taken pre-ischaemia and following ischaemia and reperfusion. Spontaneous and electrically evoked contractile activity of the circular and longitudinal smooth muscles were determined in each sample with and without the addition of dexmedetomidine. During a second experiment, tetrodotoxin was added to determine if the effect was neurogenic. We found that the circular smooth muscle (CSM) contractility was not affected by ischaemia, whereas the longitudinal smooth muscle (LSM) showed an increase in both spontaneous and induced contractile activity. The addition of dexmedetomidine caused a decrease in the spontaneous contractile activity of CSM, but an increase in that of LSM, which was not mediated by the enteric nervous system. During ischaemia, dexmedetomidine also mildly increased the electrically induced contractile activity in LSM. These results may indicate a stimulatory effect of dexmedetomidine on small intestinal contractility. However, the influence of dexmedetomidine administration on intestinal motility in vivo needs to be further investigated.