Pandemic H1N1 (pH1N1) influenza virus emerged from swine in 2009 with adequate capability to infect and transmit between people. In subsequent years it has circulated as a seasonal virus and evolved further human-adapting mutations. Mutations in the haemagglutinin (HA) stalk that increase pH stability have been associated with human adaptation and airborne transmission of pH1N1 virus. Yet, our understanding of how pH stability impacts virus/host interactions is incomplete. Here, using recombinant viruses with point mutations that alter the pH stability of pH1N1 HA, we found distinct effects on virus phenotypes in different experimental models. Increased pH sensitivity enabled virus to uncoat in endosomes more efficiently, manifesting as increased replication rate in typical continuous cell cultures under single-cycle conditions. A more acid labile HA also conferred a small reduction in sensitivity to antiviral therapeutics that act at the pH-sensitive HA fusion step. Conversely, in primary human airway epithelium cultured at air-liquid interface, increased pH sensitivity attenuated multicycle viral replication, by compromising virus survival in the extracellular microenvironment. In a mouse model of influenza pathogenicity, there was an optimum HA activation pH and viruses with either more or less pH stable HA were less virulent. Opposing pressures inside and outside the host cell that determine pH stability may influence zoonotic potential. The distinct effects that changes in pH stability exert on viral phenotypes underscore the importance of using the most appropriate systems for assessing virus titre and fitness, which has implications for vaccine manufacture, antiviral drug development and pandemic risk assessment.ImportanceThe pH stability of the haemagglutinin surface protein varies between different influenza strains and subtypes and can affect the virus' ability to replicate and transmit. Here, we demonstrate a delicate balance the virus strikes within and without the target cell. We show that a pH-stable haemagglutinin enables a human influenza virus to replicate more effectively in human airway cells and mouse lungs by facilitating virus survival in the extracellular environment of the upper respiratory tract. Conversely after entering target cells, being more pH-stable confers relative disadvantage, resulting in less efficient delivery of the viral genome to the host cell nucleus. Since the balance we describe will be affected differently in different host environments, it may restrict virus' ability to cross species. In addition, our findings imply that different influenza viruses may show variation in how well they are controlled by antiviral strategies targeting pH-dependent steps in the virus replication cycle.