We have shown that non-typeable Haemophilus influenzae (NTHI) resists killing by antimicrobial peptides (APs). A mutant defective in expression of the sap (sensitivity to antimicrobial peptides) gene cluster product SapA is sensitive to killing by APs and is significantly attenuated in its ability to survive in a chinchilla model of otitis media compared with the parent strain. In NTHI, SapA is believed to function as the periplasmic solute binding protein of an ABC transporter. Here, we demonstrated that recombinant chinchilla beta defensin-1 specifically interacted with recombinant SapA and that AP exposure increased expression of the sap operon. We further demonstrated that the putative Sap transporter ATPase protein, SapD, was required for AP resistance as well as potassium uptake in NTHI strain 86-028NP. Loss of SapD additionally abrogated NTHI survival in vivo. Complementation of the sapD mutation restored the ability to grow in potassium-limited medium, resistance to AP-mediated killing and survival in vivo. Collectively, these data support a mechanism of Sap system-mediated resistance to APs that depends on Sap-dependent transport of APs and a Sap-dependent restoration of potassium homeostasis. Thus, NTHI required a functional Sap system to mediate bacterial survival and pathogenesis in vivo.