Bone repair in horses implies invasive surgeries and increased cost. Research on musculoskeletal disorders therapy in horses includes cell-based therapy with mesenchymal stromal cells (MSCs). Mesenchymal stromal cells can be obtained from bone marrow (BMMSCs). Unfortunately, BMMSCs have limited cell replication . The objective of this study was to develop a biologically immortalized equine stem cell line derived from bone marrow, with unlimited proliferation and the ability to differentiate into bone cells. Equine BMMSCs were transfected and immortalized with human telomerase reverse transcriptase (hTERT) gene. Cell passages from equine immortal BMMSCs were characterized by the presence of stemness CD markers and expression of multi-potent differentiation genes (OCT-4, SOX2, and NANOG). Equine immortal BMMSCs were incubated in osteogenic medium and bone cell differentiation was determined by alkaline phosphatase and von Kossa staining, and osteogenic gene expression (osteocalcin, Runx2, and osterix). Telomerase activity was determined by telomeric repeat amplification technique. Results showed that equine immortal BMMSCs were able to replicate up to passage 50 and maintain stem cell characteristics by the presence of CD90 and expression of multi-potent genes. Equine immortal BMMSCs were able to differentiate into bone cells, which was confirmed by the positive osteogenic staining and gene expression. Equine BMMSCs were successfully immortalized and maintained characteristics of stem cells and readily differentiated into osteogenic cells. Extending the life span of equine BMMSCs by transfection of the hTERT gene will revolutionize the clinical use of MSCs by making them available to orthopedic surgeons "off the shelf."