AbstractReconstruction of injured bone remains challenging in the clinic owing to the lack of suitable bone grafts. The utilization of PAI‐1 transfected‐conditioned media (P‐CM) has demonstrated its ability to facilitate the differentiation process of mesenchymal stem cells (MSCs), potentially serving as a crucial mediator in tissue regeneration. This research endeavored to explore the therapeutic potential of P‐CM concerning the differentiation of human bone marrow mesenchymal stem cells (hBMSCs). To assess new bone formation, a rat calvaria critical defect model was employed, while in vitro experiments involved the use of the alizarin Red‐S mineral induction test. In the rat calvaria critical defect model, P‐CM treatment resulted in significan new bone formation. In vitro, P‐CM treated hBMSCs displayed robust osteogenesis compared to the control group, as demonstrated by the mineral induction test using alizarin Red‐S. P‐CM with hydroxyapatite/β‐tricalcium phosphate/fibrin gel treatment significantly exhibited new bone formation, and the expression of osteogenic associated markers was enhanced in the P‐CM‐treated group. In conclusion, results demonstrate that P‐CM treatment significantly enhanced the osteogenic differantiation efficiency and new bone formation, thus could be used as an ideal therapeutic biomolecule for constructing bone‐specific implants, especially for orthopedic and dental applications.