N-terminal acetylation is a major posttranslational modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), NatA through NatF. Although N-terminal acetylation modulates diverse protein functions, little is known about its roles in virus replication. We found that NatB, which comprises NAA20 and NAA25, is involved in the shutoff activity of influenza virus PA-X. The shutoff activity of PA-X was suppressed in NatB-deficient cells, and PA-X mutants that are not acetylated by NatB showed reduced shutoff activities. We also evaluated the importance of N-terminal acetylation of PA, because PA-X shares its N-terminal sequence with PA. Viral polymerase activity was reduced in NatB-deficient cells. Moreover, mutant PAs that are not acetylated by NatB lost their function in the viral polymerase complex. Taken together, our findings demonstrate that N-terminal acetylation is required for the shutoff activity of PA-X and for viral polymerase activity.