Thymic atrophy has been described as a consequence of infection by several pathogens including highly pathogenic avian influenza virus and is induced through diverse mechanisms. However, whether influenza A(H1N1)pdm09 infection induces thymic atrophy and the mechanisms underlying this process have not been completely elucidated. Our results show that severe infection of influenza A(H1N1)pdm09 led to progressive thymic atrophy and CD4+ CD8+ double-positive (DP) T-cells depletion due to apoptosis. The viruses were present in thymus, where they activated thymic innate CD8(+)CD44(hi) single-positive (SP) thymocytes to secrete a large amount of IFN-γ. Milder thymic atrophy was observed in innate CD8+ T-cell-deficient mice (C57BL/6J). Neutralization of IFN-γ could significantly rescue the atrophy, but peramivir treatment did not significantly alleviate thymic atrophy. In this study, we demonstrated that thymic innate CD8(+)CD44(hi) SP T-cells have critical roles in influenza A(H1N1)pdm09 infection-induced thymic atrophy through secreting IFN-γ. This exceptional mechanism might serve as a target for the prevention and treatment of thymic atrophy induced by influenza A(H1N1)pdm09.