Repair of bone defects presents a serious clinical challenge as it is difficult to restore bone function and regenerate bone loss. In the present study, the effects of lentivirus‑mediated transfection of bone marrow mesenchymal stem cells (BMSCs) with microRNA (miR)‑26a on bone regeneration were investigated in a mouse bone defect repair model. Marker of proliferation Ki67 (Ki67) staining was employed to detect the cell proliferation capacity and alkaline phosphatase (ALP) staining was used to investigate osteogenic differentiation. A mouse model of cranial bone defects was established. β‑tricalcium phosphate biomaterials co‑cultured with the transfected BMSCs were implanted into the defect areas of mouse models. Micro‑computed tomography, and hematoxylin and eosin and toluidine blue staining, were used to detect bone regeneration in the defect areas and the degradation of scaffolds. miR‑26a expression, and the mRNA and protein expression of osteogenesis‑associated cytokines, were detected using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Separated and cultured BMSCs highly expressed CD29 and CD105, but not CD34 and CD45, as determined by flow cytometry. miR‑26a expression and the positive cell rate of Ki67 and ALP staining in BMSCs transfected with pLVTHM‑miR‑26a were increased. The BMSC and negative control‑transfected BMSC groups exhibited increased bone regeneration in the defect areas, increased bone volume of newly formed bones, and elevated mRNA and protein expression of runt‑related transcription factor 2 (Runx2) and osteocalcin (OC), compared with the blank group. However, the miR‑26a‑transfected BMSC group exhibited further increases in bone regeneration and the volume of newly formed bones, and further elevations of the mRNA and protein expression levels of Runx2 and OC. The present findings demonstrated that lentivirus‑mediated modification of BMSCs enhanced bone regeneration during the repair of cranial bone defects in mice.