This study represents two different large-scale proteomic experiments analyzing the antibiotic response and the mechanisms of production of β-lactamases in the nosocomial pathogen Stenotrophomonas maltophilia. Two-dimensional gel electrophoresis on the cytoplasmic protein fraction, together with iTRAQ® differential labeling and 2-D liquid chromatographic separation (2D-LC) MS/MS on the enriched membrane protein fraction, revealed 73 proteins with a change in abundance upon imipenem challenge. These proteins belong to several different functional pathways. We observe an increase in β-lactamase production as well as in proteins important for their function in the periplasm. The up-regulation of the L1 and L2 β-lactamases, along with their activator LysR transcriptional factor AmpR, is linked to an increase in proteins responsible for peptidoglycan remodeling and stress response. The interesting identification of an increase in abundance after treatment of the two-component GGDEF signaling protein and an integral membrane sensor signal transduction histidine kinase, indicates that induction of the β-lactamases is not restricted to the ampR-ampD-ampG pathway. This is the first proteomic study in S. maltophilia upon imipenem stimulation to further unravel the cellular adaptation resulting in β-lactamase production.