Objective:To package the recombinant adenovirus containing HIV-1 Vif gene by using AdEasy system and detect the effect of Vif protein expression on the latent infection and lytic replication of KSHV. Methods: The fragment of Vif gene from expression vector pCI-neo-Vif was cloned into the shuttle plasmid pAdTrack-CMV. With the digested recombinant plasmid pAdTrack-Vif and the backbone plasmid pAdEasy-1, the homologous recombination took place in the E.coli BJ5183,and the recombinant adenovirus plasmid was generated. The recombination adenoviruses were packaged and amplified in the AD293 cells. Then the virus titer was checked by observing the expression of GFP. After BCBL-1 cells were infected by the recombinant adenovirus with 1 MOI, the mRNA transcripts and protein expression of Vif in BCBL-1 cells were detected by RT-PCR and Western blot. Then, the expressions of KSHV ORF26 mRNA and vIL-6 protein were detected by Real-time PCR and Western blot, respectively. Results: The recombinant adenovirus carrying HIV-1 Vif was packaged successfully with the virus titer of 2.5×108 TU/ml. After infected with adenovirus, more than 80% BCBL-1 cells could express GFP, and the exact band of Vif was detectable by RT-PCR and Western blot. Moreover, the expression of KSHV ORF26 mRNA and vIL-6 protein were down-regulated by Vif protein. Conclusion: The recombinant adenovirus carrying HIV-1 Vif was packaged successfully,and Vif protein expressed in BCBL-1 cells could inhibit KSHV lytic replication and enhance KSHV latent infection.