Analyses of gait are frequently performed during treadmill locomotion. However, few studies have compared treadmill walking and overground locomotion with respect to gait parameters. Therefore seven different spatial and temporal gait parameters have been investigated at the same natural walking speed both on a treadmill and during overground walking in twelve adults (6 females, 6 males). Treadmill walking was recorded with an infrared motion-analysis system. For analysis of overground walking the method described by Brinckmann combined with video-analysis was used. In treadmill walking the cadence was significantly enhanced (5%) while the stride length was significantly reduced (4%) compared to overground walking. This difference was due to shortened stance and double-support phases in treadmill walking while the swing phase of the walking cycle had increased. Further, step width and foot angles were significantly larger during treadmill locomotion. These results indicate that in treadmill walking gait parameters may be specifically adapted to achieve a more protective gait pattern. Larger step widths and foot angles could be explained by maintenance of the dynamic equilibrium due to unsteadyness in treadmill walking. These results are important for the discussion regarding a similarity between treadmill walking and natural gait.