Objective: To construct the lentiviral vector containing tumor suppressor gene WTX (Wilms tumor gene on the X chromosome) and the colon cancer cell line stably transduced with this WTX-containing vector (Lovo/WTX-EGFP), so as to provide a useful tool for studying the role of WTX in colon cancer. Methods: The lentiviral vector pLV.Ex3d.null-EF1A>WTX>IRES/EGFP was constructed by Gateway technology, which was screened and identified by colony PCR. After that, it was co-transfected into the 293FT cells with three helper plasmids that were PLV/helper-SL3, PLV/helper-SL4 and PLV/helper-SL5 to package lentivirus and then the viral titer was determined under fluorescence microscope. Finally, the human colon cancer Lovo cells were transduced with the WTX-containing lentiviral vector to obtain the Lovo/WTX-EGFP cell line with stable expression of WTX gene through several subcultures by repeated colony picking. Results: The lentiviral vector pLV.Ex3d.null-EF1A>WTX>IRES/EGFP constructed by Gateway technology was completely and correctly identified. The distinct green fluorescence was seen under fluorescence microscope 48 h after virus packaging and the virus titer was 5×107 TU/mL. The vector was successfully transduced into Lovo cells as evidenced by the significantly increased WTX expression level determined by both qPCR and Western blot was obviously higher than cells without transducting. The Lovo/WTX-EGFP colon cancer cell line with stable transduction of WTX containing vector was established by repeated colony picking and subcultures. Conclusion: Through Gateway technology, the WTX-containing lentiviral vector can be successfully constructed and colon cancer Lovo/WTX-EGFP cell line can be stably transduced by this WTX-containing lentiviral vector. It may provide an experimental basis for studying the role of WTX in colon cancer.