Shuttle run, or side-step cutting, is a common warm-up exercise for athletes. Anterior cruciate ligament (ACL) injury was reported to occur during the single-limb support landing, such as shuttle run. There is little attention about the speed effect on lower extremity (L/E) biomechanics during shuttle run. The purpose of this study was to investigate the L/E biomechanics at two different speeds during shuttle run. A rate of 125 bpm was defined as the slow speed in this study, and 140 bpm as the fast speed. 6 young athletes without history of L/E illness were recruited. Helen Hayes marker set was adopted, and the motion capture system was used to detect the trajectories of the passive markers. Ground reaction force (GRF) was synchronously measured by a force plate. The period of foot contact with force plate was divided into weight acceptance phase and pushing phase. The results showed the significant higher vertical GRF at fast speed in the pushing phase (p=0.04). Less knee flexion angle was also demonstrated at the rate of 140 bpm. Higher GRF and less knee flexion were reported as risk factors for ACL injury. Therefore, speed effect on L/E biomechanics during shuttle run should be taken into consideration to prevent ACL injury.