Objective:To establish human embryonic stem cell (hESC)lines labeled with red fluorescent protein (RFP). Methods:The RFP expression vector pEF1α-DsRed-Express2 was transfected into hESCs by optimized nucleofection method. Alkaline phosphatase (AP) staining,RT-PCR,immunofluorescent staining and embryoid body (EB) formation were performed to compare the characteristics of hESCs before and after nucleofection. Results:AP staining of RFP-hESCs was positive. The expression of Oct4,Sox2,Klf4 and Nanog of hESCs were no difference before and after nucleofection (P > 0.05). RFP-hESCs could differentiate into the three germ layers and could stably passage over 30 generations. Conclusion:hESC cell lines stably expressing RFP has been successfully established in this study. RFP does not affect the pluripotency properties of hESCs. The cell lines can be used as seed cells for subsequent research.