Abstract： Objective To explore the expression of lentivirus- mediated Isl1 gene in human mesenchymal stem cells (hMSC) and to detect the expressions of its downstream genes Nkx2.5 and Flk- 1.Methods Lentiviral expression vector 2K7puro/EF1α-Isl1 was constructed with multi-site Gateway vector construction technique (promotor EF1α driving Isl1 gene expression). After packaging in 293FT cells, the lentivirus was then transfected into hMSC. The Isl1-positive hMSC were screened by resistance to puromycin.Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were then used to detect the Isl1 mRNA expression in hMSC within 1-6 weeks and protein expression within 1-4 weeks after transfection,respectively. Results Lentiviral vector EF1α-Isl1 was successfully constructed, and mRNA and protein expressions of Isl1 gene in hMSC were detected after transfection. Notably, the mRNA expression of Isl1 increased over time, which was higher at week 3 (0.65±0.14) compared to weeks 1 and 2 (0.36±0.09 and 0.37±0.05, both P＜0.05) and remained stable after week 3. In the absence of inducer, expression of Isl1 downstream gene Nkx2.5 but not Flk-1 was detected in Isl1-positive hMSC. Conclusion Long-term stable expression profile of Isl1 genes may be obtained by transfection into hMSC through lentiviral vectors, which can also result in expression of downstream Nkx2.5 gene in absence of inducer.