The objective was to demonstrate walking locomotor pattern alterations after co-administration of acepromazine and morphine in horses. Six mature horses receiving four different treatments were used. Treatments consisted of a single dose of saline solution, 0.2 mg/kg bwt of morphine hydrochloride, 0.02 mg/kg bwt of acepromazine maleate, and a combination of 0.2 mg/kg bwt of morphine hydrochloride with 0.02 mg/kg bwt of acepromazine maleate. A three-dimensional accelerometric device was used to collect data. Walking tests were performed 10 min prior to injection, and then at 5, 10, 15, and 20 min after the injection, and then every 10 min for 3 h. Eight variables were calculated including stride kinematic, coordination, and energetic parameters; moreover ground-to-lip distance (GLD), as a tranquilization parameter, was also measured. A significant interaction was observed in all the variables studied but regularity, mediolateral power, the propulsive part of the power, and the GLD. An evident counteraction of the effects caused by both, opioids and phenothiazines, in the gait pattern was observed. The co-administration of acepromazine and morphine could allow a safe opiate administration while minimizing the possible central nervous system (CNS) excitation and reducing potential locomotor adverse effects.