To counteract host antiviral responses, influenza A virus triggers a global reduction of cellular gene expression, a process termed “host shutoff.” A key effector of influenza A virus host shutoff is the viral endoribonuclease PA-X, which degrades host mRNAs. While many of the molecular determinants of PA-X activity remain unknown, a previous study found that N-terminal acetylation of PA-X is required for its host shutoff activity upon ectopic expression. However, it remains unclear how this co-translational modification promotes PA-X activity. Here, we report that PA-X N-terminal acetylation has two functions—it promotes nuclear localization but is also needed directly for host shutoff. Moreover, these two functions can be separated based on whether acetylation occurs on the first amino acid, the initiator methionine, or the second amino acid following initiator methionine excision. Modification at either site is sufficient to ensure PA-X localization to the nucleus, whereas N-terminal acetylation of the initiator methionine is specifically required for normal PA-X host shutoff activity. We also demonstrate that PA-X N-terminal acetylation is needed for its activity during infection. Our studies thus uncover a multifaceted role for PA-X N-terminal acetylation in the regulation of this important immunomodulatory factor.