The goal of this study was to compare treadmill walking with overground walking in healthy subjects with no known gait disorders. Nineteen subjects were tested, where each subject walked on a split-belt instrumented treadmill as well as over a smooth, flat surface. Comparisons between walking conditions were made for temporal gait parameters such as step length and cadence, leg kinematics, joint moments and powers, and muscle activity. Overall, very few differences were found in temporal gait parameters or leg kinematics between treadmill and overground walking. Conversely, sagittal plane joint moments were found to be quite different, where during treadmill walking trials, subjects demonstrated less dorsiflexor moments, less knee extensor moments, and greater hip extensor moments. Joint powers in the sagittal plane were found to be similar at the ankle but quite different at the knee and hip joints. Differences in muscle activity were observed between the two walking modalities, particularly in the tibialis anterior throughout stance, and in the hamstrings, vastus medialis and adductor longus during swing. While differences were observed in muscle activation patterns, joint moments and joint powers between the two walking modalities, the overall patterns in these behaviors were quite similar. From a therapeutic perspective, this suggests that training individuals with neurological injuries on a treadmill appears to be justified.