To better understand physiological and pathological movement patterns in the equine thoracolumbar spine, investigation of the biomechanics on a segmental level requires a constant moment. A constant moment along the spinal column means that the same torque acts on each vertebral segment, allowing the range of motion of different segments to be compared. The aims of this study were to investigate the range of motion of the equine thoracolumbar spine in horses with and without spinal pathology and to examine whether the pressure between the spinous processes depends on the direction of the applied moment. Thoracolumbar spine specimens (T8-L4) of 23 horses were mounted in a custom-made mechanical test rig to investigate spinal biomechanics during lateral bending, axial rotation, flexion and extension using computed tomographic imaging. Results were compared between horses with spondylosis, overriding spinous processes and specimens free of gross pathology. The interspinous space pressure was additionally determined using a foil sensor. The median lateral bending between T9 and L3 was 3.7°-4.1° (IQR 5.4°-8.0°). Maximum rotational movement with inconsistent coupled motion was observed at T9-T16 (p < 0.05). The dorsoventral range of motion was greatest in segments T9-T11 (p < 0.05). Spondylosis and overriding spinous processes restricted spinal mobility, depending on the severity of the condition. There was no significant difference in interspinous pressure during motion (p = 0.54). The biomechanical study confirmed that the range of motion of intervertebral joints depends on the anatomical position of the joint and the direction of the moment applied. Restricted mobility was evident in the presence of different grades of overriding spinous processes or spondylosis. A better understanding of equine spinal biomechanics in horses with spinal pathology facilitates individual rehabilitation.