The HA (hemagglutinin) of influenza virus must be activated by proteolysis before the virus can become infectious. Previous studies have indicated that HA cleavage is driven by membrane-bound or extracellular serine proteases in the respiratory tract. However, there is still uncertainty as to which proteases are critical for activating the HA of seasonal influenza A viruses (IAV) in humans. This study focuses on human KLK1 and KLK5, two of the 15 serine proteases known as the kallikrein-related peptidases (KLK). We find that their mRNA expression in primary human bronchial cells is stimulated by an IAV infection. Both enzymes cleaved recombinant HA from several strains of H1 and/or H3 virus subtypes in vitro, but only KLK5 promoted the infectivity of A/PR/8/34 (H1N1) and A/Scotland/20/74 (H3N2) virions in MDCK cells. We assessed the ability of treated viruses to initiate influenza in mice. Nasal instillation of only KLK5-treated virus resulted in weight loss and lethal outcome. The secretion of this protease in the human lower respiratory tract is enhanced during influenza. Moreover, we show that pretreatment of airway secretions by a KLK5-selective inhibitor significantly reduced activation of influenza A/Scotland/20/74 virions, providing further evidence of its importance. Differently, increased KLK1 secretion appeared to be associated with the recruitment of inflammatory cells in human airways regardless the origin of inflammation. Thus, our findings point to the involvement of KLK5 in the proteolytic activation and spread of seasonal influenza viruses in humans.IMPORTANCE Influenza A viruses (IAV) cause acute infection of the respiratory tract that affects millions of people during seasonal outbreaks every year. Cleavage of the hemagglutinin precursor by host proteases is a critical step in the life cycle of these viruses. Consequently, host proteases that activate HA can be considered as promising targets for the development of new antivirals. However, the specific proteases that activate seasonal influenza viruses, especially H3N2 viruses, in the human respiratory tract have remain undefined despite many years of work. Here we demonstrate that the secreted, extracellular protease KLK5 (kallikrein-related peptidase 5) is efficient to promote infectivity of H3N2 IAV in vitro and in vivo Furthermore, we found that its secretion was selectively enhanced in the human lower respiratory tract during a seasonal outbreak dominated by a H3N2 virus. Collectively, our data support the clinical relevance of this protease in human influenza pathogenesis.