This study evaluated the ability of the native semitendinosus tendon to regenerate after harvest for anterior cruciate ligament (ACL) reconstruction in a rabbit model. The histological and biomechanical properties of the regenerate tendons also were studied. At 16 and 28 weeks post-harvesting of the semitendinosus tendon, the regenerate tissue was evaluated by magnetic resonance imaging, gross and histologic pathologic specimens, and biomechanical evaluation. Ten of 10 specimens at 16 weeks and 10 of 10 specimens at 28 weeks demonstrated regeneration of the semitendinosus tendon, with biomechanical strength 23% of normal tendon at 16 weeks and 62% of normal tendon at 28 weeks. The results of this study support regeneration of the semitendinosus tendon to its osseous insertion on the proximal tibia after harvesting for ACL reconstruction.