The myotendinous junction (MTJ) is a specialized structure in the musculotendinous system, where force is transmitted from muscle to tendon. Animal models have shown that the MTJ takes form of tendon finger-like processes merging with muscle tissue. The human MTJ is largely unknown and has never been described in three dimensions (3D). The aim of this study was to describe the ultrastructure of the human MTJ and render 3D reconstructions. Fourteen subjects (age 25 ± 3 years) with isolated injury of the anterior cruciate ligament (ACL), scheduled for reconstruction with a semitendinosus/gracilis graft were included. Semitendinosus and gracilis tendons were stripped as grafts for the ACL reconstruction. The MTJ was isolated from the grafts and prepared for transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy. It was possible to isolate recognizable MTJ tissue from all 14 patients. TEM images displayed similarities to observations in animals: Sarcolemmal evaginations observed as finger-like processes from the tendon and endomysium surrounding the muscle fibers, with myofilaments extending from the final Z-line of the muscle fiber merging with the tendon tissue. The 3D reconstruction revealed that tendon made ridge-like protrusions, which interdigitiated with groove-like indentations in the muscle cell.