In our previous studies, we developed a marker-based 3D motion capture system to evaluate step kinematics during behaviors such as climbing and novel environment exploration under various pharmacological treatments. In the present study, we aimed to test whether the step kinematics of mice is affected during treadmill running at different speeds and under various treatments. We used adult male C57BL/6 mice in a within-subject, randomized design. We recorded voluntary running behavior on a treadmill in mice treated with vehicle (1:1:18 ethanol, Kolliphor, saline), CP55,940 (0.3 mg/kg) or harmaline (20 mg/kg, i.p. 30 min pretreatment). Mice were implanted with permanent markers located at strategic locations: hips, shoulder blades, hindlimb knees, and ankles. A high-speed, high-resolution 3D motion capture system (Qualisys) was used to track movement (3D trajectories and velocity of markers) during voluntary running. Every mouse was recorded for 30 s running at speeds ranging from 1 m/min to 40 m/min, doubled with each trial until failure. Harmaline, at 20 mg/kg induces strong whole-body tremor and significantly inhibited the ability of mice to run on a treadmill. The maximum speed achieved was significantly decreased by harmaline and CP55,940. During treadmill running, ankle swings had significantly higher mean and maximum speeds than during vertical climbing or exploration tasks. Moreover, CP55,940 reduced the height of the ankle swing during treadmill running, similarly as it does during climbing. In conclusion, marker-based 3D motion capture is a precise method to quantify motor impairments and disruption of step kinematics in mice.