Interaction between HIV-1 Vif and host factor CBFβ leads to the assembly of the Vif-Cul5-EloB/C ubiquitin ligase (E3 complex). By inducing the formation of E3 complex, Vif depletes host APOBEC3 restriction factors and promotes HIV-1 infection. In addition, Vif is known to arrest host cells at G2/M phase (G2 arrest), benefiting HIV-1 replication and contributing to the depletion of CD4+ T cells. However, whether CBFβ is also involved in Vif-induced cell cycle arrest remains unclear. In the present study, we report that CBFβ is an essential factor for Vif-induced G2 arrest. Reducing endogenous CBFβ expression significantly compromised Vif's potency in cell cycle regulation. In addition, tests with CBFβ and Vif mutants indicated that Vif-CBFβ interaction is crucial for Vif to induce G2 arrest. Furthermore, suppressors against Vif-hijacked E3 complex or proteasome-mediated proteolysis also abolished Vif's ability to cause G2 arrest. In general, our data indicated that Vif induces G2 arrest through depletion of a yet-unknown cellular factor, where the involvement of CBFβ is essential. On the other hand, our data also suggested that, antiviral drugs targeting the Vif-CBFβ interaction have the potential to abolish Vif's ability to cause APOBEC3 degradation as well as G2 arrest in host cells, thus reducing both HIV-1 replication and Vif-induced CD4+ T-cell depletion.