Trans-tibial amputees are advised to walk as much as able people to achieve healthy and independent life. However, they usually have difficulties in doing so. Previous researches only included data from a few steps when studying the gait of amputees. Walking over a long distance was rarely examined. The objective of this study was to investigate the changes in spatial-temporal, kinetic and kinematic gait parameters of trans-tibial amputees after long-distance walking. Six male unilateral trans-tibial amputees performed two sessions of 30-min walking on a level treadmill at their self-selected comfortable speed. Gait analysis was undertaken over-ground: (1) before walking, (2) after the 1st walking session and (3) after the 2nd walking session. After the long-distance walking, changes in spatial-temporal gait parameters were small and insignificant. However, the sound side ankle rocker progression and push-off were significantly reduced. This was due to the fatigue of the sound side plantar flexors and was compensated by the greater effort in the prosthetic side. The prosthetic side knee joint showed significantly increased flexion and moment during loading response to facilitate the anterior rotation of the prosthetic shank. The prosthetic side hip extensors also provided more power at terminal stance to facilitate propulsion. Endurance training of the sound side plantar flexors, and improvements in the prosthetic design to assist anterior rotation of the prosthetic shank should improve long-distance walking in trans-tibial amputees.