Aggregation can be selectively induced by aggregation-prone regions (APRs) contained in the target proteins. Aggregation-inducing antimicrobial peptides (Pept-ins) contain sequences homologous to APRs of target proteins and exert their bactericidal effect by causing aggregation of a large number of proteins. To better understand the mechanism of action of Pept-ins and the resistance mechanisms, we analyzed the phenotypic, lipidomic, and transcriptomic as well as genotypic changes in laboratory-derived Pept-in-resistant E. coli mutator cells. The analysis showed that the Pept-in resistance mechanism is dominated by a decreased Pept-in uptake, in both laboratory-derived mutator cells and clinical isolates. Our data indicate that Pept-in uptake involves an electrostatic attraction between the Pept-in and the bacterial membrane and follows a complex mechanism potentially involving many transporters. Furthermore, it seems more challenging for bacteria to become resistant toward Pept-ins that are less dependent on electrostatic attraction for uptake, suggesting that future Pept-ins should be selected for this property.