AbstractSerine proteases have been implicated as key drivers and facilitators of lung cancer malignancy, and while these proteins represent straightforward targets for therapeutic inhibitors, identification of optimal points for intervention has been complicated by the complex networks in which these enzymes function. Here we implicate a signaling pathway consisting of PRSS3/mesotrypsin and kallikrein-related peptidase 5 (KLK5) in lung adenocarcinoma malignancy. We show that elevated PRSS3/mesotrypsin expression is prognostic for poor outcome for patients with lung adenocarcinoma, and that genetic or pharmacologic targeting of PRSS3/mesotrypsin reduces lung adenocarcinoma cell invasiveness and proliferation. We further show that genetic targeting of KLK5, a known target of PRSS3/mesotrypsin, phenocopies the effect of PRSS3/mesotrypsin knockdown, and also that elevated expression of KLK5 is similarly prognostic for outcome in lung adenocarcinoma. Finally, we use transcriptional profiling experiments to show that PRSS3/mesotrypsin and KLK5 control a common malignancy-promoting pathway. These experiments implicate a potential PRSS3/mesotrypsin-KLK5 signaling module in lung adenocarcinoma and reveal the potential therapeutic benefit of selectively targeting these pathways.