The zinc-finger antiviral protein (ZAP) is a host factor that inhibits the replication of certain viruses, including murine leukemia virus and Sindbis virus by destabilizing viral mRNA in the cytoplasm. ZAP binds to specific viral mRNAs and recruits cellular RNA degradation machinery to degrade the RNA. Identifying ZAP-interacting proteins provides a viable strategy to uncover the mechanism by which ZAP inhibits viral replication. In the present study, we developed a method to search for proteins interacting with ZAP. Lysates of ZAP-expressing cells were subjected to glycerol gradient centrifugation. Fractions co-migrating with ZAP were collected, followed by immunoprecipitation of ZAP. Proteins co-immunoprecipitated with ZAP were identified by mass spectrometric analysis. By this method, PR65A, a structural subunit of PP2A, was identified as a putative ZAP-interacting protein. Coimmunoprecipitation assays confirmed the interaction between PR65A and ZAP in an RNA-independent manner. Downregulation of PR65A reduced the antiviral activity of ZAP. We conclude that PR65A interacts with ZAP and is required for the optimal antiviral activity of ZAP.