Large and retracted rotator cuff tendon tears fail to repair or retear after surgical intervention. This study attempted to develop novel tissue-engineering approaches using tenocyte-seeded bioscaffolds for tendon reconstruction of massive rotator cuff tendon defect in rabbits. Porcine small intestine submucosa (Restore) and type I/III collagen bioscaffold (ACI-Maix) were chosen as bioscaffold carriers for autologous tenocytes. Biological characterization of autologous tenocytes was conducted before the implantation. The tenocyte-seeded bioscaffolds were implanted as interposition grafts to reconstruct massive rotator cuff tendon defects in rabbits. In situ reimplantation of the autologous rotator cuff tendon, excised during defect creation, served as a positive control. Histological outcomes were analyzed and semi-quantitatively graded at 4 and 8 weeks after surgery. At 4 weeks, both tenocyte-seeded bioscaffolds displayed inflammatory reaction similar to bioscaffold-only cuff reconstruction, and the histological grading were inferior to control repair. However, at 8 weeks, inflammatory reaction of both tenocyte-seeded bioscaffolds were dramatically less than with bioscaffold alone. In addition, bioscaffolds seeded with tenocytes generated a histological appearance similar to that of the positive control. The implantation of autologous tenocytes on collagen-based bioscaffolds results in better rotator cuff tendon healing and remodeling than with the implantation of bioscaffold alone.