Objective: To construct a lentiviral vector expressing HIV-1 Tat and identify its expression in 293T cells. Methods: The gene fragment of HIV-1 Tat101 was subcloned to lentiviral transfer vector pHAGE-CMV-MCS-IZsGreen, which was named pHAGE-Tat. Then the constructed pHAGE-Tat was used to co-transfect the packing 293T cells, together with the packaging plasmids pMD2.G and psPAX2. The packaged viral particles designated LV-Tat were used to infect the 293T cells and the viral titer was calculated. The expression of HIV-1 Tat in 293T cells was confirmed using RT-PCR and western blot. Results: The recombinant lentiviral vector was successfully constructed and could express HIV-1 Tat in 293T cells. The virus titer was 5.73×106 ifu/ml. Conclusion: The successfully constructed recombinant lentiviral vector makes a strong foundation for further exploring the possible role of HIV-1 Tat in the development of prostate cancer.