Inspired by the native biomineralization mechanisms of the bone matrix, we developed a growth factor-free, nanoscale-engineered silk fibroin (NESF) hydrogel to enhance in situ biomineralization and promote effective endogenous bone regeneration. By modulating SF nanofiber length to enrich acidic groups, the hydrogel acquired an increased negative surface charge, providing initial physical cues that effectively attracted inorganic ions and promoted Ca2+-PO43- nucleation, resulting in a 6.09-fold enhancement in biomineralization. These intrinsic chemical modifications further boosted the biological activity of the NESF hydrogel by promoting the adhesion and proliferation of BMSCs, and facilitated osteogenic differentiation by elevating intracellular Ca2+ influx and activating the MAPK/ERK signaling pathway.