Using soft tissue grafts for anterior cruciate ligament (ACL) reconstruction, insertion site healing plays a crucial role in the long-term fate of the graft. It has been shown in an experimental animal study that using a soft tissue graft and anatomic graft fixation, a direct ligamentous insertion alike the native ACL developed 24 weeks postoperatively. Yet there are no reports on the long-term insertion site healing of anatomically fixed soft tissue grafts. The objective of this study was to evaluate graft insertion site healing, the intra-tunnel fate of the graft and its osseous replacement 2 years after ACL reconstruction in sheep. The left ACLs of six sheep were replaced by an autologous flexor tendon split graft and anatomically fixed with biodegradable poly-(D, L-lactide) interference screws. Animals received polychromic sequential labeling at different points in time to determine bone apposition per period. For evaluation of the insertion site healing and intra-tunnel changes, MRI scans were taken in vivo. Following sacrifice, radiographic imaging, conventional histology and fluorescence microscopy was undertaken. Most of the specimens showed a wide direct ligamentous insertion. It showed patterns alike the direct ligament insertion seen in intact ACLs. The intra-tunnel part of the graft had completely lost its tendon-like structure and in two cases, it was separated from the graft insertion by a thick bony layer. The biodegradable interference screw was fully degraded in all specimens. Ossification of the former drill tunnels was intense, showing only partial-length tunnel remnants in one femoral and three tibial specimens. As the graft heals to the joint surface and the aperture site is closed with soft tissue, mechanical stress of the intra-tunnel part of the graft is eliminated and the bone tunnel is protected from synovial fluid, resulting in osseous bridging of the tunnel aperture site, accelerated intra-tunnel graft resorption and its osseous replacement.