Regeneration of the gradient structure of the tendon-to-bone interface (TBI) is a crucial goal after rotator cuff repair. The purpose of this study was to investigate the efficacy of a biomimetic hydroxyapatite-gradient scaffold (HA-G scaffold) isolated from adipose tissue (AD) with umbilical cord derived mesenchymal stem cells (UC MSCs) on the regeneration of the structure of the TBI by analyzing the histological and biomechanical changes in a rat repair model. As a result, the HA-G scaffold had progressively increased numbers of hydroxyapatite (HA) particles from the tendon to the bone phase. After seeding UC MSCs to the scaffold, specific matrices, such as collagen, glycoaminoglycan, and calcium, were synthesized with respect to the HA density. In a rat repair model, compared to the repair group, the UC MSCs seeded HA-G scaffold group had improved collagen organization and cartilage formation by 52% at 8 weeks and 262.96% at 4 weeks respectively. Moreover, ultimate failure load also increased by 30.71% at 4 weeks in the UC MSCs seeded HA-G scaffold group compared to the repair group. Especially, the improved values were comparable to values in normal tissue. This study demonstrated that HA-G scaffold isolated from AD induced UC MSCs to form tendon, cartilage and bone matrices similar to the TBI structure according to the HA density. Furthermore, UC MSC-seeded HA-G scaffold regenerated the TBI of the rotator cuff in a rat repair model in terms of histological and biomechanical properties similar to the normal TBI. Statement of Significance We found specific extracellular matrix (ECM) formation in the biomimetic-hydroxyapatite-gradient-scaffold (HA-G-scaffold) in vitro as well as improved histological and biomechanical results of repaired rotator cuff after the scaffold implantation in a rat model. This study has four strengths; An ECM scaffold derived from human adipose tissue; only one-layer used for a gradient scaffold not a multilayer used to mimic the unique structure of the gradient tendon-to-bone-interface (TBI) of the rotator cuff; UC-MSCs as a new cell source for TBI regeneration; and the UC-MSCs synthesized specific matrices with respect to the HA density without any other stimuli. This study suggested that the UC-MSC seeded HA-G-scaffold could be used as a promising strategy for the regeneration of rotator cuff tears.Copyright © 2020. Published by Elsevier Ltd.