BACKGROUND: Stem cell-conditioned medium (CM) has been increasingly used in regenerative medicine. However, its effect on graft-host integration after anterior cruciate ligament (ACL) reconstruction (ACLR) remains unclear. PURPOSE: To examine the effect of human bone marrow stem cell (hBMSC)-CM on graft-bone integration and graft midsubstance ligamentization in a rat model of ACLR. STUDY DESIGN: Controlled laboratory study. METHODS: CM was obtained from the supernatant of commercially available hBMSCs in serum-free Dulbecco's modified Eagle medium (DMEM). In a rat model of an ACL injury, isometric ACLR was performed. Three groups were established: CM injection group (CM; n = 40), control injection group (CI; n = 40) with serum-free DMEM injections, and no injection group (NI; n = 40). An intra-articular injection was performed weekly. Micro-computed tomography was conducted at 2, 4, and 8 weeks postoperatively. Histological and biomechanical analyses were conducted at 4 and 8 weeks postoperatively. The NIH3T3 fibroblast was utilized as a model in vitro to examine the effect of CM using the cell counting kit-8 (CCK-8) assay and immunofluorescence staining of Ki-67, α-smooth muscle actin (α-SMA), and collagen 1 (Col 1). RESULTS: At 4 and 8 weeks, the femoral and tibial bone tunnel areas as well as the interface between the graft and host bone were smaller, while the bone volume/total volume ratio was higher, in the CM group. Sharpey-like fibers formed at 8 weeks in the CM group. At 4 and 8 weeks, more Col 1 was noticed in the CM group than in the NI group (both < .001) or CI group (both < .001). Immunohistochemically, the α-SMA-positive area was up-regulated at the graft-bone interface at 4 weeks ( < .001) and declined at 8 weeks ( < .001) in the CM group compared with the other 2 groups. At the midsubstance, α-SMA expression decreased from 4 to 8 weeks in all groups and was significantly lower in the CM group than in the NI group ( < .01) or CI group ( < .05) at 8 weeks. The CCK-8 assay showed that CM increased NIH3T3 viability ( < .001) and the level of Ki-67 ( < .05), α-SMA ( < .001), and Col 1 ( < .001) in CM-educated NIH3T3 cells. CONCLUSION: hBMSC-CM accelerates graft-bone incorporation and midsubstance ligamentization and enhances the proliferation, differentiation, and collagen synthesis of fibroblasts. CLINICAL RELEVANCE: Graft-host integration is essential after ACLR. The current study identified a novel agent, that is, hBMSC-CM, as a candidate for promoting integration.