Stem cells have potential utility in wound therapy, however the benefits are often limited due to cell injury from shear stress during injection and poor retention at the wound site. Here, shear-thinning silk nanofiber hydrogels were used to load bone marrow derived mesenchymal stem cells (BMSCs) and inject into wound sites to optimize cell retention and accelerate wound healing. The BMSCs in the silk nanofiber hydrogels maintained stemness better than the cells cultured on plates, and the expression of wound healing-related genes was significantly higher in the hydrogels with higher silk concentrations (2 wt%). The silk nanofibers physically prevented migration of BMSCs from the deposition site in the wound bed. In addition to faster wound healing, these BMSC-loaded hydrogels mediated angiogenesis and inflammation and improved collagen deposition and hair follicle regeneration in vivo in rats. Considering that these silk nanofiber hydrogels were successfully used here as carriers for stem cells to accelerate wound healing, further study for skin regeneration may be warranted.