Tissue-engineered bone substitutes are frequently used to repair bone defects. Adipose-derived stem cells (ASCs) are a promising source of cells for repairing bone tissue, however, insufficient osteogenic potency remains the main obstacle for their application. The present study aimed to enhance the osteogenic potency of ASCs by transfection of microRNA (miR)‑26a, a novel osteogenic and angiogenic promoting miRNA. An inverted fluorescence microscope was used to observe transfection efficiency, while a scanning electron microscope was used to detect morphological alterations. Cell proliferation was monitored continuously for 7 days using a Cell Counting kit‑8 assay. Osteogenic differentiation was determined by reverse transcription quantitative polymerase chain reaction, alkaline phosphatase (ALP) staining, collagen secretion and extracellular matrix (ECM) mineralization. ASCs were incorporated with a porous hydroxyapatite (HA) scaffold to create a novel tissue‑engineered bone substitute and inserted into the critical tibia defect of rats. New bone formation was evaluated by hematoxylin and eosin and Masson's trichrome staining. The results demonstrated that miR‑26a was successfully delivered into the cytoplasm, while the morphology and proliferation of ASCs were not significantly altered. Osteogenic‑associated genes were markedly upregulated and ALP production, collagen secretion and ECM mineralization were all increased following transfection of miR‑26a. Histological evaluation demonstrated that the modified cells accompanied with a porous HA scaffold markedly promoted new bone formation within the defective area. In conclusion, miR‑26a transfection significantly improved the osteogenic potency of ASCs suggesting that modified ASCs incorporated with a HA scaffold may be used as a potential bone substitute.