Methotrexate (MTX) is commonly used in cancer chemotherapy to treat childhood leukaemia and osteosarcoma. Although MTX chemotherapy improves the population of cancer survivors, the prevalence of chronic bone-related complications has increased. Reduced bone formation (osteogenesis) and increased marrow fat formation (adipogenesis) have been observed through a "switch-like" change in commitment of bone marrow stromal cells (BMSCs) following MTX treatment. However, the underlying molecular mechanisms of this bone/fat switch are not fully elucidated. MicroRNAs (miRNAs) participate in regulating BMSC differentiation by targeting osteogenesis/adipogenesis-related genes. Here, we found miR-6315 and miR-542-3p were differentially expressed in bone samples from MTX-treated rats. Target prediction tool and miRNA-mRNA network analyses indicated that Smad2 and Smurf2/sFRP-1 might be the direct target of miR-6315 and miR-542-3p, respectively. Subsequent luciferase assays confirmed the predictions. Additionally, in vitro cell models were applied to determine the potential roles of these miRNAs on osteogenic and adipogenic differentiation. Results suggest that miRNA agmoir supplement (miR-6315/miR-542-3p) enhanced osteogenesis, characterized by a significant increase in expression of osteogenesis markers RUNX2, ALP, OCN and OSX. On the other hand, miRNA agomir treatment inhibited adipogenesis and lipid droplet accumulation. Signalling pathway analyses demonstrated that miR-6315 can regulate bone/fat formation through the Smad2/TGF-β signalling and miR-542-3p can modulate bone/fat formation via the Wnt/β-catenin signalling. These findings may increase our understanding of how MTX damages the bone and may provide a foundation for further studies investigating potential bone damage biomarkers or therapeutic targets for patients who receive MTX chemotherapy.