The HIV-1 Rev (Regulator of Expression of Virion) protein, an RNA-binding protein essential for viral replication, is imported into the host cell nucleus by human Importin β (Impβ). Rev is phosphorylated in vivo on serine residues by a nuclear kinase. In this study, we introduced glutamate substitution mutations that mimic phosphorylation at serine positions previously identified as potential phosphorylation sites and assessed their impact on the ability of Rev to bind Impβ in thermal shift, gel shift, and fluorescence polarization assays. Phosphomimetic mutations introduced in either the N-terminal tail, helical hairpin domain or C-terminal domain of Rev had a small but reproducible stabilizing effect on the Impβ/Rev complex. Moreover, the mutation of Rev residue Ser56, which localizes to one face of the helical hairpin domain, had a greater stabilizing effect than that of Ser54 located on the opposite face, suggesting that the helical hairpin orients its Ser56-containing face towards Impβ. Taken together, our results suggest that phosphorylation can significantly modulate the ability of Rev to associate with Impβ.