The aim of the present study was to explore the feasibility of lentiviral-mediated sodium iodide symporter (NIS) gene delivery for monitoring bone marrow-derived mesenchymal stem cell (BMSC) transplantation into the infarcted myocardium. For this purpose, we constructed a lentiviral vector (Lv-EF1α-NIS-IRES-EGFP) expressing NIS and enhanced green fluorescent protein (EGFP), and introduced it into BMSCs at different multiplicities of infection (MOI). The expression of EGFP was observed under a fluorescence microscope. Iodine uptake and the inhibition of iodine uptake by sodium perchlorate (NaClO4) in the Lv-EF1α-NIS-IRES-EGFP?treated BMSCs were dynamically monitored in vitro. The Lv-EF1α-NIS-IRES-EGFP?treated BMSCs were transplanted into the infarcted myocardium of Sprague-Dawley rats, and 99mTc99g (Tc, technetium; 99m indicates that technetium is at its excited stage; 99g indicates the atomic weight of technetium) micro-single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging was performed in vivo 1 week following transplantation. The isolated BMSCs successfully differentiated into adipocytes and osteoblasts. The BMSCs were positive for the cell surface markers, CD105, CD29 and CD90, and negative for CD14, CD34 and CD45. Lv-EF1α-NIS-IRES-EGFP was efficiently transfected into the BMSCs. RT-qPCR and western blot analysis confirmed that the BMSCs expressed high protein and mRNA levels of NIS by day 7 following infection, and NIS expression remained at a consistent level from day 14 to 21. In the Lv-EF1α-NIS-IRES-EGFP?treated BMSCs, the accumulation of iodine-125 (125I) was observed in vitro and was successfully monitored by 99mTc99g micro-SPECT/CT imaging at 1 week following transplantation. These results suggest that lentiviral vectors are powerful vehicles for studying gene delivery in BMSCs. It is feasible to use lentiviral vectors to deliver an NIS gene for the non-invasive monitoring of BMSC transplantation and survival in the infarcted myocardium in vivo.