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Enthesis injury repair remains a huge challenge because of the unique biomolecular composition, microstructure, and mechanics in the interfacial region. Surgical reconstruction often creates new bone–scaffold interfaces with mismatched properties, resulting in poor osseointegration. To mimic the natural interface tissue structures and properties, we fabricated a nanofibrous scaffold with gradient mineral coating based on 10 × simulated body fluid (SBF) and silk fibroin (SF). We then characterized the physicochemical properties of the scaffold and evaluated its biological functions both in vitro and in vivo. The results showed that different areas of SF nanofibrous scaffold had varying levels of mineralization with disparate mechanical properties and had different effects on bone marrow mesenchymal stem cell growth and differentiation. Furthermore, the gradient scaffolds exhibited an enhancement of integration in the tendon-to-bone interface with a higher ultimate load and more fibrocartilage-like tissue formation. These findings demonstrate that the silk-based nanofibrous scaffold with gradient mineral coating can regulate the formation of interfacial tissue and has the potential to be applied in interface tissue engineering.