Known and novel cellular proteins and RNP partners of Vif were identified by mass spectrometry in Vif-Flag–transfected HeLa cells. Using mass spectrometry, we identified two different groups of proteins with which Vif interacts: the first group contained a number of RNA-associated proteins (eg, ribonucleoproteins hnRNP U and hnRNP L), RNA helicases (DHX9 and Gu), and some pre-mRNA splicing factors (PRP9, spliceosomal protein). The second group included two cell-cycle regulatory proteins, Cdk9 and Brd4. The criteria for protein identification were more than 2 peptides matched and a mascot score > 35.