BACKGROUND Our previous experiments indicated that bone marrow mesenchymal stem cells of rhesus monkey (RhBMSCs) have a low proliferative ability with a finite life span, which will hamper their application in biomedical research. Establishing an immortalized RhBMSC lineage might solve the problem. METHODS RhBMSCs isolated from the bone marrow of rhesus monkeys using density gradient centrifugation were purified using adherence separation. Then, the cells were steadily transfected by plasmid containing human telomerase reverse transcriptase gene (pCI-neo-hTERT). We analyzed expression of hTERT, proliferation, phenotype (SH-2, SH-3, SB-10, CD29, CD34, CD45, and HLA-DR), differentiation toward osteogenic lineage, karyotype, and tumorigenesis of transfected cells. RESULTS After transfection, the RhBMSCs proliferated vigorously, undergoing more than 50 population doublings (PDs). Apoptotic rate of transfected RhBMSCs at PD40 was only 4.5%, versus untransfected RhBMSCs at PD15, which was more than 33.5%. Compared with normal RhBMSC, the life span of transfected RhBMSCs was prolonged, retaining similar morphology, karyotype, and potential to differentiate into an osteogenic lineage. More than 99% of transfected RhBMSCs were positive for stem cell markers, including SH-2, SH-3, SB-10, and CD29, and negative for CD34, CD45, and HLA-DR. Furthermore, the transfected cell line was benign in nude mice tumor formation. CONCLUSIONS Our results demonstrated that hTERT gene had been transfected into RhBMSCs. The transfected RhBMSCs proliferated vigorously. Phenotype, differentiation, and karyotype of transfected RhBMSC showed no significant difference from untransfected cells. The transfected RhBMSCs are a potential cell source for transplantation as well as tissue engineering.