Although miRNAs have been implicated in the osteogenic differentiation of stem cells, their role in bone repair and reconstruction in tissue-engineered bone grafts remains unclear. We previously reported that microRNA (miR)-26a-5p inhibited the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs), and that antimiR-26a-5p exerted the opposite effect. In the present study, the role of miR-26a-5p- and antimiR-26a-5p-modified ADSCs combined with biphasic calcium phosphate (BCP) scaffolds was evaluated in a rat femur defect model. The aim of the present study was to improve the understanding of the role of miR-26a-5p in bone regeneration
in vivo
, as well as to provide a new method to optimize the osteogenic ability of BCPs. ADSCs were infected with Lv-miR-26a-5p, Lv-miR-NC, Lv-antimiR-26a-5p or Lv-antimiR-NC respectively, and then combined with BCP scaffolds to repair rat femoral defects. Using X-rays, micro-computed tomography and histology at 2, 4, and 8 weeks postoperatively, the quantity and rate of bone regeneration were analyzed, revealing that they were the highest in animals treated with antimiR-26a-5p and the lowest in the miR-26a-5p treatment group. The expression levels of osteocalcin, collagen I, Runt-related transcription factor 2, Wnt family member 5A and calmodulin-dependent protein kinase II proteins were positively correlated with the bone formation rate. Taken together, the present results demonstrated that miR-26a-5p inhibited bone formation while antimiR-26a-5p accelerated bone formation via the Wnt/Ca
2+
signaling pathway. Therefore, antimiR-26a-5p-modified ADSCs combined with BCP scaffolds may be used to construct an effective tissue-engineering bone graft for bone repair and reconstruction.