Huanglian-Houpo drug combination (HHDC) is a classical traditional Chinese medicine that has been effectively used to treat seasonal colds and flu. However, no systematic studies of the effects of HHDC on H1N1 influenza infection and the associated mechanisms have been reported. The aim of the present study was to determine the anti-H1N1 influenza effects of HHDC and explore the underlying mechanisms. A mouse model of H1N1-induced pneumonia was established and the mice were treated with HHDC (4, 8 and 16 g/kg) for 5 days after viral challenge. The antiviral effects of HHDC and the underlying mechanisms in the mice were investigated and evaluated with respect to inflammation, oxidative stress and Toll-like receptor (TLR)/myeloid differentiation factor (MyD88)/nuclear factor (NF)-κB signaling pathways. HHDC provided significant protection against weight loss and reduced the H1N1 viral load in the lungs. In addition, HHDC significantly decreased the lung index and increased the spleen and thymus indices of the H1N1-infected mice. HHDC also significantly ameliorated the histopathological changes of pneumonia, decreased serum levels of the cytokines interleukin (IL)-6, tumor necrosis factor-α and interferon-γ, and increased the serum level of IL-2. Moreover, HHDC significantly increased the levels of the antioxidant factors superoxide dismutase and glutathione, and reduced the serum level of nitric oxide. Furthermore, the mRNA and protein expression levels of TLR3, TLR7, MyD88, NF-κB p65 and tumor necrosis factor receptor-associated factor 3 in the lung tissues were significantly decreased by HHDC. These findings suggest that HHDC directly inhibited H1N1 infection in vivo and exerted a therapeutic effect on influenza-induced pneumonia in mice by modulating cytokines, antioxidant factors and TLR/MyD88/NF-κB signaling pathways. Copyright: © Zhang et al.