Bone mesenchymal stem cells (BMSCs) are the most commonly investigated progenitor cells in bone tissue engineering for treating severe bone defects. Strategies for regulating BMSC differentiation fate have received wide attention, in which redox homeostasis plays an important role due to the change in energy metabolism during stem cell differentiation. In the present study, it was observed that autophagic activity was induced along with BMSC osteogenic differentiation and subsequently regulated reactive oxygen species (ROS) generation and the level of osteogenesis. Furthermore, it was also observed that neuropeptide substance P (SP) administration could enhance the autophagic activity in rat BMSCs via the AMPK and mTOR pathways, as well as decreasing ROS generation and promoting osteogenic differentiation. Inhibition of autophagic activity by 3‑MA reversed the effects of SP on ROS and osteogenic levels. The present results indicated that autophagic activity participated in the regulation of differentiation fate of BMSCs and SP could promote osteogenic differentiation by activating autophagy, providing a more precise biological mechanism for its application in bone tissue engineering.