In the field of bone tissue engineering, scaffold materials play a central role in restoring and regenerating the physiological function of the skeletal system. Mechanical signals in the cellular microenvironment significantly influence cellular behavior, and the selection of an appropriate scaffold matrix is crucial in guiding precursor cells towards their desired cell fate. The aim of this study is to investigate the impact of matrix stiffness on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and whether matrix stiffness affects BMSCs osteogenic differentiation in an autophagy-dependent manner. BMSCs were co-cultured with transglutaminase (TG)-Gelatin (Gel) matrices of varying stiffness, and subsequently, osteogenic markers in each experimental group were evaluated. Additionally, the levels of autophagy within the cells were quantitatively measured. Furthermore, inhibitor experiments were conducted to gain deeper insights into potential mechanisms. We observed that within a certain range of stiffness, autophagy levels increased with the augmentation of matrix stiffness, leading to an elevation in the levels of osteogenesis-related molecules.