Objective To establish a culture system of bone marrow mesenchymal stem cells (BMMSCs) in vitro in a mouse spinal muscular atrophy (SMA) model, study the antisense oligonucleotide (ASO) influence of its biological characteristics, and establish a reliable simulation tool with the SMA model for the in depth study of the pathogenesis of SMA and drug screening. Methods The marrow of the femur and humeral bones of four-day-old SMA mice were harvested. The surface markers of BMMSCs were identified by immunofluorescence. The survival motor neuron gene 2 (SMN 2) exon7 inclusion level and survival motor neuron (SMN) protein expression were detected by RT-PCR and Western Blot. The cell proliferation and apoptosis were detected by an EDU kit and TUNEL kit. Results The BMMSCs isolated and cultured in vitro from SMA mice showed adherent growth and the ability to passage. P3 generation BMMSCs were identified by cellular immunofluorescence, in which the positive markers of CD44 and CD29 were highly expressed, and the negative markers of CD34 and CD45 expression were low. SMN2 exon7 inclusion and SMN protein expression increased significantly after ASO transfected the cells, and the proliferation was significantly promoted, while the number of gemi bodies within the nucleus also increased. Conclusions A BMMSC in vitro culture system for SMA mice was successfully established to promote the inclusion of BMMSC SMN2 exon7 and the expression of SMN protein via ASO verification as a new tool cell for the study of SMN2 -related regulatory mechanisms and drug screening.