•The polypeptide OP 3-4 promoted bone regeneration of mandibular defect.•OP 3-4 improved osteogenic differentiation of bone marrow mesenchymal stem cells.•OP 3-4 promoted osteogenic differentiation by AKT/GSK3β/β-catenin pathway. The aims of this study were to explore: (ⅰ) the effect of the polypeptide OP 3-4 on bone regeneration in vivo; (ⅱ) the effect of OP 3-4 on osteogenic differentiation of bone marrow mesenchymal stem cells in vitro; and (ⅲ) the potential mechanism of OP 3-4 in promoting osteogenic differentiation of bone marrow mesenchymal stem cells. 30 Wistar rats (8-week, male) were randomly divided into Control group (n = 5), Hydrogel group (n = 5), and Hydrogel loaded OP 3-4 group (n = 5). Hematoxylin and eosin staining was used to evaluate the level of bone regeneration in mandibular defect. Immunohistochemistry staining was used to evaluate the expression of alkaline phosphatase, runt-related transcription factor 2, and type Ⅰ collagen. Flow cytometry was applied to identify the phenotype of bone marrow mesenchymal stem cells. Furthermore, LY294002, the inhibitor of protein kinase B, was applied to verify the role of OP 3-4 in promoting osteogenic differentiation via protein kinase B/glycogen synthase kinase 3β/β-catenin pathway through western blot. OP 3-4 promoted bone regeneration of rat mandibular defect. The expression of osteogenic differentiation related markers were increased after adding OP 3-4 to bone marrow mesenchymal stem cells. OP 3-4 promoted osteogenic differentiation of bone marrow mesenchymal stem cells via protein kinase B/glycogen synthase kinase 3β/β-catenin pathway. OP 3-4 could promote bone regeneration of mandibular defect and improve osteogenic differentiation through protein kinase B/glycogen synthase kinase 3β/β-catenin pathway.