Objective: To evaluate the differences between walking on an advanced robotic locomotion interface called the Treadport and walking overground with healthy subjects. Background: Previous studies have compared treadmill-based and overground walking in terms of gait parameters. The Treadport’s unique features including self-selected speed capability, large belt, kinesthetic force feedback, and virtual reality environment distinguish it from other locomotion interfaces and could provide a natural walking experience for the users. Method: Young, healthy subjects ( N = 17) walked 10 meters 10 times each for both overground and the Treadport environments. Comparison between walking conditions used spatiotemporal and kinematic parameters. In addition, electromyographic data was collected for five of the 17 subjects to compare muscle activity between the two conditions. Results: Gait on the Treadport was found to have no significant differences ( p > .05) with overground walking in terms of hip and knee joint angles, cadence and stride length and stride speed, and muscle activation of the four muscle groups measured. Differences ( p < .05) were observed in ankle dorsiflexion which was reduced by 2.47 ± 0.01 degrees on the Treadport. Conclusion: Walking overground and on the Treadport is highly correlated and not significantly different in 13 of 14 parameters. Application: This study suggests that the Treadport creates an environment for natural walking experience, where natural gait of users is almost preserved, with great potential to be useful for other applications, such as gait rehabilitation of individuals with walking impairments.