Semitendinosus and gracilis muscles are frequently harvested for autologous tendon grafts for cruciate ligament reconstruction. This study investigated the joint-stabilizing effects of these hamstring muscles in cases of insufficiency of the medial collateral ligament (MCL). First, both the semitendinosus and gracilis muscles can actively stabilize the joint against valgus moments in the MCL-deficient knee. Second, the stabilizing influence of these muscles decreases with an increasing knee flexion angle. Controlled laboratory study. The kinematics was examined in 10 fresh-frozen human cadaveric knees using a robotic/universal force moment sensor system and an optical tracking system. The knee kinematics under 5- and 10-N·m valgus moments were determined in the different flexion angles of the (1) MCL-intact and (2) MCL-deficient knee using the following simulated muscle loads: (1) 0-N (idle) load, (2) 200-N semitendinosus (ST) load, and (3) 280-N (200/80-N) combined semitendinosus/gracilis (STGT) load. Cutting the MCL increased the valgus angle under all tested conditions and angles compared with the MCL-intact knee by 4.3° to 8.1° for the 5-N·m valgus moment and 6.5° to 11.9° for the 10-N·m valgus moment (P < .01). The applied 200-N simulated ST load reduced the valgus angle significantly at 0°, 10°, 20°, and 30° of flexion under 5- and 10-N·m valgus moments (P < .05). At 0°, 10°, and 20° of flexion, these values were close to those for the MCL-intact joint under the respective moments (both P > .05). The combined 280-N simulated STGT load significantly reduced the valgus angle in 0°, 10°, and 20° of flexion under 5- and 10-N·m valgus moments (P < .05) to values near those for the intact joint (5 N·m: 0°, 10°; 10 N·m: 0°, 10°, 20°; P > .05). In 60° and 90° of flexion, ST and STGT loads did not decrease the resulting valgus angle of the MCL-deficient knee without hamstring loads (P > .05 vs deficient; P = .0001 vs intact). In this human cadaveric study, semitendinosus and gracilis muscles successfully stabilize valgus moments applied to the MCL-insufficient knee when the knee is near extension. In the valgus-unstable knee, these data suggest that the hamstring muscles should be preserved in (multi-) ligament surgery when possible.