Hypothesis: Permeation of macromolecular drugs across biological plasma membranes is a major challenge in drug delivery. Cationic cell-penetrating peptides (CPPs) are attractive functional excipient candidates 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 facilitate 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 techniques. The experimental findings were supported by molecular dynamics (MD) simulations, demonstrating 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 displayed upon CPP interaction. Our results demonstrate that the impact exerted by the CPP on the membrane is notably affected by positioning and especially the degree of lipidation, which might influence the properties of CPPs as functional excipients.