Purpose To observing biological characterization of MSCs labelled with SPIO and Fth1 gene.To explore the optimal concentration range of labeling mesenchymal stem cells with SPIO. To estimate feasibility, effectiveness and security of this method that establishing transgenic rat mesenchymal stem cells carring Fth1. And to provide tracing method for research on stem cell therapy for clinical study in vivo. Methods MSCs were isolated and cultured primarily by whole bone marrow plastic adherence method. The MSCs were incubated with SPIO-PLL complexes at the range of concentrations (25, 50, 75,100μg/ml SPIO, and 0.75μg/ml PLL) for different time (48 hours to 28 days); infected MSCs with Fth1 gene for different time. Cell viability was tested through Trypan blue staining; cell proliferation was evaluated by MTT growth test. MSCs labelled with SPIO and Fth1 were scanned by MRI in different periods to investigate signals of cells. Results In experimental groups of MSCs labelled with SPIO at the concentrations (25, 50 μg/ml SPIO, and 0.75μg/ml PLL) and Fth1 gene, there was no significant statistical difference on cell proliferative activity and cell viability compared with the controlled group. After culturing for 28 days, there was no MRI signal in MSCs labelled with 50μg/ml SPIO. Fth1-MSCs cultured for 48 hours and 28 days, cell suspension showed T2 low signal intensity on MRI imaging, signal intensity of the two periods had no significant statistical difference. Compared with MSCs labelled with 50μg/ml SPIO, signal intensity of Fth1-MSCs had no statistical difference in the period of 48 hours; but there was significant statistical difference in the period of 28 days(P=0.0012). Conclusions SPIO at the concentration range of 25~50μg/ml and FTH1 gene can label and transfect MSCs safely and effectively, do not affect cell biological characteristics of phenotype, viability and proliferation. Fth1-MSCs had genetic stability, showed T2 low signal by MRI.