Bone marrow mesenchymal stem cells (BM‑MSCs) are pluripotent stem cells that are regarded as ideal resources for the reconstruction of tissues and organs. The Tibetan mastiff is a breed of domesticated Chinese native dog that is well‑adjusted to the high‑altitude environments of Tibet. To the best of our knowledge, the biological characterization and multi‑lineage differentiation of Tibetan mastiff BM‑MSCs have not been reported previously. Therefore, the present study aimed to investigate the biological characteristics and therapeutic potential of Tibetan mastiff BM‑MSCs. A cell culture system was constructed and cells were cultured to 23 passages in vitro. Growth curves and colony formation studies suggested that BM‑MSCs had a high self‑renewal capacity and that their proliferation rate declined with age. Karyotype analysis demonstrated that BM‑MSCs were diploid and genetically stable. Semi‑quantitative polymerase chain reaction analysis revealed that BM‑MSCs positively expressed cluster of differentiation (CD)73, CD90, CD105, CD166 and vimentin, although they were negative for the endothelial cell marker CD31. Additionally, immunofluorescence staining revealed that the cells expressed CD29, CD44, CD90, CD105 and vimentin. Flow cytometric analysis revealed that the rates of positive expression of vimentin, CD44, CD90 and CD105 were all >97%. BM‑MSCs were able to differentiate into adipocytes, osteoblasts, cartilage cells, hepatocytes and functional insulin‑secreting cells. In conclusion, Tibetan mastiff BM‑MSCs may be purified successfully using a whole bone marrow culture method. The findings of the current study suggested important potential applications of BM‑MSCs as a source for regenerative therapies.