There is a paucity of evidence on the effect that rider asymmetry has on equine locomotion. The aim of this study was to evaluate the effect of rider asymmetry on equine locomotion by using a novel approach to induce rider asymmetry. Ten nonlame horses were recruited for this study. Joint center markers were used to capture 2D kinematics (Quintic Biomechanics) of the horse and rider and horses were equipped with seven inertial sensors positioned at the fifth (T5) and eighteenth (T18) thoracic vertebrae, third lumbar (L3) vertebra, tubera sacrale (TS), and left and right tubera coxae. Rider asymmetry was induced by shortening the ventral aspect of one stirrup by 5 cm. Kinematic data were compared between conditions using a mixed model with the horse defined as a random factor and stirrup condition (symmetrical stirrups and asymmetrical stirrups) and direction (inside and outside) defined as fixed factors. Data from riders where the right stirrup was shortened were mirrored to reflect a left stirrup being shortened. To determine differences between conditions, a significance of P ≤. 05 was set. On the rein with the shortened stirrup on the outside: an increase in lateral bending range of motion (ROM) at T5 (P = .003), L3 (P = .04), and TS (P = .02), an increase in mediolateral displacement at T5 (P = .04), T18 (P = .04), and L3 (0.03) were found. An increase in maximum fetlock extension was apparent for both the front (P = .01) and hind limb (P = .04) on the contralateral side to the shortened stirrup; for the asymmetrical stirrup condition on the rein with the shortened stirrup on the inside: an increase in flexion-extension ROM at T5 (P = .03) and L3 (P = .04), axial rotation at T5 (P = .05), and lateral bending of T5 (P = .03), L3 (P = .04), and TS (P = .02). Asymmetric rider position appears to have an effect on the kinematics of the thoracolumbar spine. These findings warrant further investigation to understand the long-term impact this may have on equine locomotor health.