The purpose of the present study was to investigate the effect of the horse's laterality on the symmetry of rein tension in right-handed riders. Eleven right-handed riders rode both a right-lateralized (RL) and a left-lateralized (LL) horse. Rein tension was measured during three circles of walk, trot and canter and four walk–halt transitions in each direction. Tensions were recorded continuously using a rein tension meter. The LL horse was ridden with significantly stronger mean tension in the left rein than in the right rein (1.5 vs. 1.4 kg; P = 0.0352). Significantly more tension was applied to the outside rein in a clockwise (1.4 vs. 1.2 kg; P = 0.0202), but not in a counterclockwise, direction (1.3 vs. 1.2 kg; P = 0.49). Less minimum tension (0.06 vs. 0.29 kg) and greater maximum (6.4 vs. 3.9 kg) and range of tension (6.3 vs. 3.6 kg) occurred in the left rein of the RL horse (P < 0.0001) and the right rein of the LL horse (0.37 vs. 0.08 kg, 4.8 vs. 7.4 kg, 4.3 vs. 7.3 kg respectively; P < 0.0001). The results of the present study indicate that the different utilization of both reins is likely to be influenced by the laterality of both horse and rider. These findings may have important implications for equine training, since consistency of reinforcement is an important factor for equine learning success.