In this paper, the design and experimental validation of a knee exoskeleton are presented. The exoskeleton can capture the negative work from the wearer’s knee motion while decreasing the muscle activities of the wearer. First, the human knee biomechanics during the normal walking is described. Then, the design of the exoskeleton is presented. The exoskeleton mainly includes a left one-way transmission mechanism, a right one-way transmission mechanism, and a front transmission mechanism. The left and right one-way transmission mechanisms are designed to capture the negative work from the wearer’s knee motion in the stance and swing phases, respectively. The front transmission mechanism is designed to transform the bidirectional rotation of the wearer’s knee joint into the generator unidirectional rotation. Additionally, the modeling and analysis of the energy harvesting of the exoskeleton is described. Finally, walking experiments are performed to validate the effectiveness of the proposed knee exoskeleton. The testing results verify that the developed knee exoskeleton can output a maximum power of 5.68 ± 0.23 W and an average power of 1.45 ± 0.13 W at a speed of 4.5 km/h in a gait cycle. The average rectus femoris and semitendinosus activities of the wearers in a gait cycle are decreased by 3.68% and 3.40%, respectively.