Summary Background The use of combinations of α2‐adrenergic agonists and opioids has been published as providing superior sedation than either drug alone. Objectives The present study aimed to compare the sedative and physiological effects of intravenous (IV) administration of dexmedetomidine alone and in combination with methadone, morphine, tramadol or pethidine in miniature donkeys. Study design Experimental, crossover Latin square, randomised, blinded study. Methods Twelve clinically healthy miniature donkeys with a mean age of 6 ± 2 (mean ± SD) years, weight of 94 ± 5 kg and height at withers of 0.80 ± 0.06 m were assigned to six IV treatments. They received either 0.9% saline, 5 μg/kg dexmedetomidine, 5 μg/kg dexmedetomidine and 0.2 mg/kg methadone, 5 μg/kg dexmedetomidine and 0.1 mg/kg morphine, 5 μg/kg dexmedetomidine and 2 mg/kg tramadol or 5 μg/kg dexmedetomidine and 1 mg/kg pethidine, with a washout period of 8 days. The degree of sedation was investigated using a numerical ranking scale of 0–3. Heart rate (HR), respiratory rate (RR), rectal temperature (RT) and intestinal motility were performed before and 5, 10, 15, 30, 45, 60, 75, 90 and 120 min after drug administration. Results Animals in dexmedetomidine or dexmedetomidine/opioid treatments were sedated for 5–60 min. Sedation was significantly higher in dexmedetomidine/opioid treatments than in dexmedetomidine at 15–30 min (p < 0.05). In all treatments, HR and RR significantly decreased from baseline at 5–75 and 30–60 min, respectively (p < 0.05). Intestinal motility was decreased in dexmedetomidine and dexmedetomidine/opioid treatments at 5–60 and 5–90 min, respectively. Compared to dexmedetomidine, intestinal motility was significantly lower in dexmedetomidine/opioid treatments at 75–90 min. Main limitations Assessment of only HR and RR are inadequate to describe the cardiorespiratory effects of dexmedetomidine/opioids. Conclusions The use of combinations of dexmedetomidine/opioids would be considered for superior sedation for 15–30 min after administration in miniature donkeys. No significant differences were detected between opioids in combination with dexmedetomidine in miniature donkeys.