The influenza A virus protein PA-X comprises an N-terminal PA region and a C-terminal PA-X-specific region. PA-X suppresses host gene expression, termed shutoff, via mRNA cleavage. Although the endonuclease active site in the N-terminal PA region of PA-X and basic amino acids in the C-terminal PA-X-specific region are known to be important for PA-X shutoff activity, other amino acids may also play a role. Here, we used yeast to identify novel amino acids of PA-X that are important for PA-X shutoff activity. Unlike wild-type PA-X, most PA-X mutants predominantly localized in the cytoplasm, indicating that these mutations decreased the shutoff activity of PA-X by affecting PA-X translocation to the nucleus. Mapping of the identified amino acids onto the N-terminal structure of PA revealed that some of them likely contribute to the formation of the endonuclease active site of PA.