OBJECTIVE: This study aims to assess the effect and mechanism of genetically modified adipose-derived mesenchymal stem cells (ASCs) with recombinant lentiviruses mediated knockdown of miR-140-5p in ASCs' osteogenesis in vitro and atrophic nonunion rat model. MATERIALS AND METHODS: This study included 36 male adult Sprague-Dawley (SD) rats weighing 400 g to 450 g from the experimental animal facility of our university. Approval was obtained from the University Animal Care Committee before the study. Rats' ASCs were prepared and genetically modified with lentivirus (Lv)-empty (NC) or Lv-miR-140-5p-TuD (inhibitors). After that, the expressions of RUNX2 and osteocalcin (OCN) were detected in the ASCs. To confirm the mechanisms of miR-140-5p in ASCs, we predicted the target genes by bioinformatics analysis and then the target genes were verified by luciferase reporting assay. The artificial atrophic nonunion was created in the rat's femoral bone. Animals were randomly divided into three groups according to the material implanted into bone defects space: AT scaffolds (AT group, n=12), AT scaffold with Lv-NC modified (AT+ASCs+Lv-NC group, n=12), AT scaffold with the Lv-miR-140-5p-TuD modified ASCs (AT+ASCs+Lv-miR-140-5p-TuD group, n=12). After four weeks, the rats were euthanized for the following radiographic examination, histologic study and biomechanical testing. RESULTS: MiR-140-5p was down-regulated during osteogenic differentiation of ASCs, and inhibition of MiR-140-5p promoted osteogenesis of ASCs in vitro. Inhibition of MiR-140-5p promoted osteogenesis of ASCs and enhanced fracture in the atrophic nonunion rat model: AT+ ASCs+LvNC group, AT+ASCs+Lv-miR-140-5p-TuD group resulted in a better bone formation and higher BMD and BMC than AT group, while excellent bone formation and the highest BMD and BMC were observed in AT+ASCs+Lv-miR-1405p-TuD group. Both AT+ASCs+Lv-NC group and AT+ASCs+Lv-miR-140-5p-TuD group presented more mature characteristics in the micro-architecture than AT group, whereas AT+ASCs+LvmiR-140-5p-TuD group presented the highest BV/TV, Tb.Th and Tb.N as well as the lowest Tb.Sp. The peak load of the operated femur increased by 94.43% AT+ASCs+Lv-miR-140-5p-TuD group, 50.68% in AT+ASCs+Lv-NC group compared to the control AT group, respectively. The result of luciferase reporting assay showed that miR-140-5p could directly target TLR4 and BMP2. CONCLUSIONS: This study demonstrates that lentiviruses-mediated knockdown of miR-140-5p can significantly promote osteogenesis of ACSs by directly regulating its' target genes, TLR4 and BMP2, and that combined adipose scaffold with genetically modified ASCs can significantly enhance fracture-healing and bone formation in the atrophic nonunion rat model.