The dysregulation of the inflammatory microenvironment following tendon injury significantly hinders regeneration. In this study, we developed an all-silk-derived functional scaffold (rKL@MPs-ASF) by integrating silk fibroin (SF) microspheres (MPs) loaded with the anti-inflammatory protein recombinant α-Klotho (rKL) into a biomimetic aligned SF (ASF) scaffold. This scaffold is designed to regulate the inflammatory microenvironment and facilitate tendon regeneration. Proteomic analysis revealed that rKL preserves the tenogenic differentiation potential of tendon stem/progenitor cells (TSPCs) by mitigating the oxidative stress response in a tumor necrosis factor-alpha-induced inflammatory microenvironment in vitro. The rKL@MPs-ASF scaffold demonstrated good drug loading/release capabilities and biocompatibility in vitro. In a rat full-thickness Achilles tendon defect model, the rKL@MPs-ASF scaffold reduced inflammatory cells infiltration and promoted fibroblast infiltration compared to the PBS@MPs-ASF group at 4 weeks post-operation. At 8 weeks post-operation, rKL@MPs-ASF-treated tendons showed increased collagen fiber deposition and reduced heterogeneous ossification, facilitating tendon regeneration and functional recovery. In conclusion, this all-silk-derived functional system creates a conducive microenvironment for tendon regeneration. STATEMENT OF SIGNIFICANCE: Regulation of the inflammatory microenvironment plays a crucial role in modulating the differentiation of TSPCs, thereby promoting tendon tissue regeneration. In this study, we demonstrate that rKL effectively preserves the tenogenic differentiation potential of TSPCs by mitigating oxidative stress within an inflammatory microenvironment. We developed an innovative, all-silk-based functional system (rKL@MPs-ASF), which integrates rKL-loaded SF MPs into an aligned silk fibroin scaffold. This system enables the controlled release of rKL, thereby modulating inflammation, promoting collagen fiber deposition, inhibiting heterotopic ossification, and ultimately improving tendon regeneration and functional recovery. Our findings highlight the potential of the rKL@MPs-ASF system, which combines structural and biological properties with a versatile drug-delivery platform, as a promising strategy for enhancing tendon repair and regenerative outcomes.