BACKGROUND: 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). HYPOTHESES: 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. STUDY DESIGN: Controlled laboratory study. METHODS: 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. RESULTS: 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). CONCLUSION: 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. CLINICAL RELEVANCE: In the valgus-unstable knee, these data suggest that the hamstring muscles should be preserved in (multi-) ligament surgery when possible.