The aim of this study was to identify phenotypic changes in a laboratory-derived strain of ertapenem-resistant Escherichia coli (Ec-ERT) when compared to its susceptible parent strain (Ec-WT). In both strains, we assessed both the effects of ertapenem via time-kill curves and the occurrence of cross resistance with other beta-lactams. The strains were compared based on growth pattern, biochemical-physiological profile and changes in the subproteome using 2D-DIGE followed by MALDI-TOF/TOF MS. To assess virulence, we employed a murine model of intraperitoneal infection in which we investigated the invasiveness of both strains. Growth persistence of the laboratory-derived resistant strain was observed via the time-kill curve assay, but cross resistance was not observed for other beta-lactams. We also observed a slower growth rate and changes in the biochemical and physiological characteristics of the drug-resistant bacteria. In the resistant strain, a total of 51 protein spots were increased in abundance relative to the wild-type strain, including an outer membrane protein A, which is related to bacterial virulence. The mouse infection assay showed a higher invasiveness of the Ec-ERT strain in relation to the Ec-WT strain. In conclusion, the alterations driven by ertapenem in E. coli reinforce the idea that antimicrobial agents may interfere in several aspects of bacterial cell biology, with possible implications for host-bacteria interactions.