Background Soft tissue allografts are essential for revision and multiple ligament surgeries in the knee, where donor-site morbidity is an issue. However, the use of allografts is associated with a higher failure rate of osteointegration. Mesenchymal stem cells (MSCs) are investigated as potential agents to enhance bone tunnel and tendon healing. Purpose This study was conducted to analyze the effect of coating allografts with MSCs on the quality and rate of osteointegration at the allograft tendon and bone interface, and the biomechanical properties of these enhanced anterior cruciate ligament (ACL) grafts compared with controls. Study Design Descriptive laboratory study. Methods Bilateral ACL reconstructions using Achilles tendon allografts were performed in 36 rabbits. On 1 limb, the graft was coated with autogenous MSCs in a fibrin glue carrier, while the contralateral limb served as a control with no MSCs. The reconstructions were assessed histologically and biomechanically at 2, 4, and 8 weeks. Results At 8 weeks, histologic analysis of the controls revealed the development of mature scar tissue resembling Sharpey fibers spanning the tendon-bone interface. In contrast, the MSC-enhanced reconstructions showed a mature zone of fibrocartilage blending from bone to the allograft, strongly resembling a normal ACL insertion. On biomechanical testing, the MSC-enhanced grafts had significantly higher load-to-failure rates than controls. However, the stiffness and Young's modulus were lower in the treatment group. Conclusions The application of MSCs at the allograft tendon-bone interface during ACL reconstruction results in the development of an intervening zone of fibrocartilage. The use of MSCs to enhance allograft osteointegration is a novel method offering the potential of more physiologic and earlier healing, although further investigation must be conducted to improve the biomechanical strength. Clinical Relevance Mesenchymal stem cells can improve the biologic properties of soft tissue allograft healing. Combined with the decrease in donor-site morbidity, allografts are a viable choice for the sports medicine surgeon.