Influenza virus entry is mediated by the acidic pH-induced activation of hemagglutinin (HA) protein. Here, we investigated how a decrease in HA activation pH (an increase in acid stability) influences the properties of highly pathogenic H5N1 influenza virus in mammalian hosts. We generated isogenic A/Vietnam/1203/2004 (H5N1) viruses containing either wild-type HA protein (activation pH 6.0) or an HA2-K58I point mutation (activation pH 5.5). The VN1203-HA2-K58I virus had similar replication kinetics compared to VN1203-wild-type in MDCK and normal human bronchial epithelial cells yet reduced growth in human alveolar A549 cells, which were found to have a higher endosomal pH than MDCK cells. Wild-type and HA2-K58I viruses promoted similar morbidity and mortality in C57BL/6J mice and ferrets, and neither virus transmitted efficiently to na?ve contact cage-mate ferrets. The acid-stabilizing HA2-K58I mutation, which diminishes H5N1 replication and transmission in ducks, increased the virus load in the ferret nasal cavity early during infection while simultaneously reducing the virus load in the lungs. Overall, a single, acid-stabilizing mutation was found to enhance the growth of an H5N1 influenza virus in the mammalian upper respiratory tract yet was insufficient to enable contact transmission in ferrets in the absence of additional mutations that confer α (2, 6)-receptor binding specificity and remove a critical N-linked glycosylation site. Information provided here on the contribution of HA acid stability to H5N1 influenza virus fitness and transmissibility in mammals, in the background of a non-laboratory-adapted virus, provides essential information for surveillance and assessment of the pandemic potential of currently circulating H5N1 viruses.