The aim of this experiment is to investigate whether MSCs from Banna Minipig Inbred Line (BMI) could be immortalized by introducing SV40 large T antigen gene. MSCs were isolated from BMI and transfected with pSV3neo plasmid. Normal BMI-MSCs would apoptosis and senescence during proliferation while its population doubling (PD) number exceeded 20. However, SV40-transfected cells were immortal. As same as that of normal BMI-MSCs, transfected MSCs were positive for stem cell markers and negative for differentiated osteoblast specific marker. After cultured in osteogenesis supplement media, cbfa1 and calcium deposit on stimulated cells were enhanced obviously. There were no data to prove the tumorigenicity potential of the immortalized cells. Furthermore, histological analysis demonstrated that bone formation was initiated in the pores of HA/TCP implants loaded immortalized BMI-MSCs 7 weeks postimplantation. BMI-MSCs were immortalized by introducing SV40 large T antigen into the cells and still kept the stem cell characters and might be used as seeding cells for tissue engineering as well as stable test cells for biocompatibility of bone biomaterials.