BACKGROUND: Vascular CD34+ cells in anterior cruciate ligament (ACL) tissue have the potential for high proliferation and multilineage differentiation that can accelerate tendon-bone healing. While patient characteristics, such as age, can affect tendon-bone healing, the influence of elapsed time after injury on the healing process is unclear. HYPOTHESIS: Cells obtained during the early phase after injury will exhibit a greater tendon-bone healing potential compared with chronic phase counterparts when applied to an immunodeficient rat model of ACL reconstruction. STUDY DESIGN: Controlled laboratory study. METHODS: Adult human ACL-ruptured tissue was harvested from patients undergoing arthroscopic primary ACL reconstruction and classified into 2 groups based on the time elapsed between injury and surgery: (1) early group (≤3 months from injury) and (2) chronic group (>3 months from injury). In addition, 76 ten-week-old female immunodeficient rats underwent ACL reconstruction, followed by intracapsular administration of one of the following: (1) ACL-derived cells from the early group (n = 5), (2) ACL-derived cells from the chronic group (n = 5), or (3) phosphate-buffered saline (PBS) only (n = 5). During the 8 weeks after surgery, histological (weeks 2, 4, 8), immunohistochemical (week 2), radiographic (weeks 0, 2, 4, 8), and biomechanical (week 8) analyses were performed to evaluate tendon-bone healing. RESULTS: In the early group, the histological evaluation showed early healing, induction of endochondral ossification-like integration, and mature bone ingrowth. Micro-computed tomography showed that the tibial bone tunnels at week 4 and week 8 were significantly reduced in the early group compared with those in the chronic group and PBS group ( P < .05). Moreover, biomechanical tensile strength was significantly greater in the early group than in the other groups ( P < .05). An accelerated healing potential in the early group was further demonstrated by the enhancement of intrinsic angiogenesis/osteogenesis and human-derived vasculogenesis/osteogenesis. CONCLUSION: Compared with human ACL-derived cells obtained during the chronic phase, cells obtained during the early phase after injury have a greater tendon-bone healing potential when used in an immunodeficient rat model of ACL reconstruction. CLINICAL RELEVANCE: During ACL reconstruction surgery, transplanting ACL remnant tissue in the early phase after injury could accelerate and enhance tendon-bone healing.