The two-dimensional (2D) kinematic approach is by far the most popular technique in rat gait analysis. This is a simple inexpensive procedure, which requires only one camera to record the movement. However, maximal precision and accuracy of the kinematic values are expected when the experimental protocol includes a three-dimensional (3D) motion analysis methodology. Locomotor speed is a basic kinematic parameter that is often neglected in most studies of movement disorders and neurological diseases. Because locomotor speed can act as confounder for the interpretation of the obtained results we also focused our attention on the relation between speed and 3D hindlimb kinematics. Our experimental set-up consisted of a motion capture system with four CMOS cameras which allowed a non-invasive estimation of the instantaneous position of color markers in a 3D measurement volume. Data were recorded while rats walked at different treadmill speeds (30 vs 60 cm/s). For the first time we reported detailed kinematic data for the sagittal, coronal and transverse plane during treadmill locomotion in rats. Despite the overall time course patterns of the curves were identical, we found significant differences between values of joint angular motion at 30 and 60 cm/s at selected points of the step cycle. The adaptation to higher treadmill walking included greater joint angular excursions. The present report highlights the importance of walking speed when evaluating rat hindlimb kinematics during gait. Hopefully, this study will be useful in experimental data assessment when multiple gait abnormalities are expected to occur in all planes of motion.