Hypothesis: Permeation of macromolecular drugs across biological plasma membranes is a major chal-lenge in drug delivery. Cationic cell-penetrating peptides (CPPs) are attractive functional excipient can-didates for the delivery of macromolecules across membrane barriers, due to their membrane translocating ability. The properties of CPPs can be tailored by lipidation, a promising approach to facil-itate enhanced membrane insertion, potentially promoting increased translocation of the CPP and cargo. Experiments: To explore the impact that site and degree of lipidation have on the membrane interaction of a cationic CPP, we designed and investigated CPP conjugates with one or two fatty acid chains. Findings: Compared to the parent CPP and the single-lipidated conjugates, the double-lipidated conjugate exhibited the most pronounced membrane perturbation effects, as measured by several biophysical tech-niques. The experimental findings were supported by molecular dynamics (MD) simulations, demon-strating that all CPP conjugates interacted with the membrane by insertion of the lipid chain(s) into the core of the bilayer. Moreover, membrane-thinning effects and induced membrane curvature were dis-played upon CPP interaction. Our results demonstrate that the impact exerted by the CPP on the mem-brane is notably affected by positioning and especially the degree of lipidation, which might influence the properties of CPPs as functional excipients. (c) 2020 Elsevier Inc. All rights reserved.