Exactly why human infection of avian influenza A virus H7N9 causes more severe disease in the elderly remains elusive. In this study, we found that H7N9 PB1-F2 is a pathogenic factor in 15–18-month-old BALB/C mice (aged mice) but not in 6–8-week-old young adult mice (young mice). Recombinant influenza A virus with H7N9 PB1-F2-knockout was less pathogenic in aged mice as indicated with delayed weight loss. In contrast, survival of young mice infected with this virus was diminished. Furthermore, tissue damage, inflammation, proinflammatory cytokine and 2′3′-cGAMP production in the lung were less pronounced in infected aged mice despite no change in viral titer. cGAS is known to produce 2′3′-cGAMP to boost proinflammatory cytokine expression through STING-NF-κB signaling. We found that H7N9 PB1-F2 promoted interferon β (IFNβ) and chemokine gene expression in cultured cells through the mitochondrial DNA-cGAS-STING-NF-κB pathway. H7N9 PB1-F2 formed protein aggregate and caused mitochondrial cristae collapse, complex V-dependent electron transport dysfunction, reverse electron transfer-dependent oxidized mitochondrial DNA release to the cytoplasm and activation of cGAS-STING-NF-κB signaling. PB1-F2 N57 truncation, which is frequently observed in human circulating strains, mitigated H7N9 PB1-F2-mediated mitochondrial dysfunction and cGAS activation. In addition, we found that PB1-F2 of pathogenic avian influenza viruses triggered more robust cGAS activation than their human-adapted descendants. Our findings provide one explanation to age-dependent pathogenesis of H7N9 infection.