Influence of bone marrow-derived mesenchymal stem cells (BMMSCs) on the healing of rabbit tibial fractures and the role of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway in fracture healing were explored. Rabbit BMMSCs were isolated and cultured in vitro, and their purity was determined using flow cytometry. Rabbit fracture models were established, and injected with BMMSCs, BMMSCs + TG101348 or TG101348, with those injected with an equal volume of normal saline as control group, and the repair of fracture ends was evaluated via X-ray examination 3 weeks later. The BMMSCs isolated in vitro grew well, and flow cytometry assay results showed that the positive expression rates of cluster of differentiation (CD)90 and CD105 in cells were 99.21 and 99.56%, respectively, with no CD45 expressed. According to the results of CCK-8 assay, TG101348 lowered the proliferation level of BMMSCs, and the wound healing assay revealed that the migration ability of BMMSCs at 24 and 48 h was substantially weaker than that in control group (P<0.05). After induction of osteogenic differentiation for 3 weeks, alizarin red staining results manifested that osteogenic induction group had notably more calcium nodules than TG101348 group (P<0.05). Compared with those in control group, the protein expression levels of p-JAK2 and p-STAT3 were remarkably raised by osteogenic induction (P<0.05), but the protein expression levels of JAK2, p-JAK2 and p-STAT3 were considerably decreased by TG101348 (P<0.05). It was found through the X-ray examination that the rabbits in control group and those injected with BMMSCs recovered well, and the latter had larger external calluses at fracture ends than the former, while the fracture ends of those injected with TG101348 and BMMSCs + TG101348 were not healed completely. BMMSCs promote healing of rabbit tibial fractures through the JAK-STAT signaling pathway.