AIMS: Influenza A virus (IAV) infection markedly up-regulates ectopic trypsins in various organs, viral envelope glycoprotein processing proteases, which are pre-requisites for virus entry and multiplication. We investigated the pathological roles of trypsin up-regulation in the progression of IAV-induced myocarditis, cytokine induction, and viral replication in the hearts, and also investigated the protective effects of trypsin inhibitor on cardiac dysfunction in vivo and selective knockdown of trypsin on IAV-induced cellular damage in cardiomyoblasts. METHODS AND RESULTS: The relationship of the expression among IAV RNA, trypsins, matrix metalloproteinase (MMP)-9, MMP-2, pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumour necrosis factor-α was analysed in mice hearts and cardiomyoblasts after IAV infection. The severity of myocarditis was most noticeable during Day 6-9 post-infection, along with peak expression of viral RNA, trypsins, particularly trypsin₂, MMPs, and cytokines. Cardiac ATP levels were the lowest at Day 9. Up-regulated trypsins, viral protein, and tissue-injured loci in the myocardium were closely localized. Trypsin inhibitor aprotinin treatment in vivo and selective trypsin₁- and trypsin₂-knockdown, particularly the latter, in H9c2 cardiomyoblasts significantly suppressed viral replication, up-regulation of MMPs, and production of active MMP-9 and cytokines, resulting in marked protection against cellular damage, ATP depletion, and apoptosis. IAV infection-induced cardiac dysfunction monitored by echocardiography was improved significantly by aprotinin treatment. CONCLUSIONS: IAV-induced trypsins, particularly trypsin₂, in the myocardium trigger acute viral myocarditis through stimulation of IAV replication, proMMP-9 activation, and cytokine induction. These results suggest that up-regulation of trypsins is one of the key host pathological findings in IAV-induced myocarditis.