H9N2 influenza viruses have been circulating worldwide in multiple avian species and regularly infect pigs and humans. Recently, a novel protein PA-X, produced from ribosomal frame-shift during PA translation, is demonstrated to be an anti-virulence factor in that it moderated infection severity in pandemic 2009 H1N1, highly pathogenic avian H5N1 and 1918 H1N1 viruses. However, a similar role of PA-X in the prevalent H9N2 avian influenza viruses has not been established. In this study, we compared the virulence and cytopathogenicity H9N2 wild type virus and H9N2 PA-X deficient virus. Loss of PA-X in H9N2 virus reduced apoptosis and had marginal effect on progeny virus output in human pulmonary adenocarcinoma (A549) cells. Without PA-X, PA was less able to suppress co-expressed green fluorescence protein (GFP) in human 293T cells. Furthermore, absence of PA-X in H9N2 virus attenuated viral pathogenicity in mice which showed no mortality, reduced progeny virus production, mild to normal lung histopathology, and dampened proinflammatory cytokine and chemokine response. Therefore, unlike previously reported H1N1 and H5N1 viruses, we show that PA-X protein in H9N2 virus is a pro-virulence factor in facilitating viral pathogenicity, and that the pro- or anti-virulence role of PA-X in influenza viruses is virus-strain dependent.