Treadmills are widely used to recover walking function in the rehabilitation field for those patients with gait disorders. Nevertheless, the ultimate goal of walking function recovery is to walk on the ground rather than on the treadmill. This study aims to determine the effect of treadmill walking on gait and upper trunk movement characteristics using wearable sensors. Eight healthy male subjects are recruited to perform 420-m straight overground walking (OW) and 5 min treadmill walking (TW), wearing 3 inertial measurement units and a pair of insole sensors. In addition to common linear features, nonlinear features, which contains sample entropy, maximal Lyapunov exponent and fractal dynamic of stride intervals (detrended fluctuation analysis), are used to compare the difference between TW and OW condition. Canonical correlation analysis is also used to indicate the correlation between upper trunk movement characteristics and gait features in the aspects of spatiotemporal parameters and gait dynamic features. The experimental results show that the treadmill can cause a shorter stride length, less stride time and worsen long-range correlation of stride intervals. And the treadmill can significantly increase the stability for both gait and upper trunk, while it can significantly reduce gait regularity during swing phase. Canonical correlation analysis results show that treadmill can reduce the correlation between gait and upper trunk features. One possible interpretation of these results is that people tend to walk more cautiously to prevent the risk of falling and neglect the coordination between gait and upper trunk when walking on the treadmill. This study can provide fundamental insightful information about the effect of treadmill walking on gait and upper trunk to support future similar studies.