Antimicrobial peptides (AMPs) are widely considered as potential alternatives and/or complements to classic antibiotics. These membrane-active peptides comprise a number of classes among which the histidine-rich family is of particular interest due to potential pH-sensitive interactions with cell membranes. In this study, we investigated pH dependence of the interactions of an AMP caerin 1.1 (Cae-1), from Australian tree frogs, containing three histidine residues with model cell membranes using NMR spectroscopy and molecular dynamics simulations. Cae-1 induced greater perturbation of the lipid dynamics and water penetrations within the membrane interior in an acidic environment compared with physiological conditions. These results from the NMR as well as the molecular dynamics study, suggested that Cae-1 was mainly inserted within the lipid bilayer for both neutral and negatively charged membranes, with the charged residues pulling the water and phosphate groups inward. This indicates that Cae-1 acts via a transmembrane mechanism in bilayers of neutral and anionic phospholipid membranes, especially in acidic conditions which could be an early step in the mechanism of membrane disruption by histidine-rich AMPs.