This study investigated the effect of hamstring co-contraction with quadriceps on the kinematics of the human knee joint and the in-situ forces in the anterior cruciate ligament (ACL) during a simulated isometric extension motion of the knee. Cadaveric human knee specimens (n=10) were tested using the robotic/universal force–moment sensor (UFS) system and measurements of knee kinematics and in-situ forces in the ACL were based on reference positions on the path of passive flexion/extension motion of the knee. With an isolated 200 N quadriceps load, the knee underwent anterior and lateral tibial translation as well as internal tibial rotation with respect to the femur. Both translation and rotation increased when the knee was flexed from full extension to 30° of flexion;​ with further flexion, these motion decreased. The addition of 80 N antagonistic hamstrings load significantly reduced both anterior and lateral tibial translation as well as internal tibial rotation at knee flexion angles tested except at full extension. At 30° of flexion, the anterior tibial translation, lateral tibial translation, and internal tibial rotation were significantly reduced by 18, 46, and 30%, respectively (p<0.05). The in-situ forces in the ACL under the quadriceps load were found to increase from 27.8±9.3 N at full extension to a maximum of 44.9±13.8 N at 15° of flexion and then decrease to 10 N beyond 60° of flexion. The in-situ force at 15° was significantly higher than that at other flexion angles (p<0.05). The addition of the hamstring load of 80 N significantly reduced the in-situ forces in the ACL at 15, 30 and 60° of flexion by 30, 43, and 44%, respectively (p<0.05). These data demonstrate that maximum knee motion may not necessarily correspond to the highest in situ forces in the ACL. The data also suggest that hamstring co-contraction with quadriceps is effective in reducing excessive forces in the ACL particularly between 15 and 60° of knee flexion.