Recruiting endogenous stem cells to bone defects without stem cell transplantation and exogenous factor delivery represents a promising strategy for bone regeneration. Herein, we develop an alkaline shear-thinning micro-nanocomposite hydrogel (10-MmN), aiming to alkaline-activate endogenous TGFβ1 and achieve in situ bone regeneration. It contains polyethyleneimine (PEI)-modified gelatin, laponite nanoplatelets (LAP), a bicarbonate buffer with a pH of 10, and gelatin microspheres (MSs). PEI-modified gelatin plays a pivotal role in hydrogel fabrication. It endows the system with sufficient positive charges, and forms a shear-thinning nanocomposite matrix in the pH 10 buffer (10-mN) with negatively charged LAP via electrostatic gelation. For biological functions, the pH 10 buffer dominates alkaline activation of endogenous serum TGFβ1 to recruit rat bone marrow stem cells through the Smad pathway, followed by improved osteogenic differentiation. In addition, MSs are incorporated into 10-mN to form 10-MmN, and function as substrates to provide good attachment sites for the recruited stem cells and facilitate further their osteogenic differentiation. In a rat critical-sized calvarial defect model, 10-MmN exhibits excellent biocompatibility, biodegradability, hydrogel infusion and retention in bone defects with flexible shapes and active bleeding. Importantly, it repairs ~95% of the defect areas in 3 months by recruiting TGFβR2+ and CD90+CD146+ stem cells, and promoting cell proliferation, osteogenic differentiation and bone formation. The present study provides a biomaterial-based strategy to regulate alkalinity in bone defects for the initiation of endogenous TGFβ signaling, which can be extended to treat other diseases.