beta-Lactamases represent the major resistance mechanism of gram-negative bacteria against beta-lactam antibiotics. The amino acid sequences of these proteins vary widely, but all are located in the periplasm of bacteria. In this study, we investigated the translocation mechanism of representative beta-lactamases in an Escherichia coli model. N-terminal signal sequence analyses, antibiotic activity assay, and direct measurement of translocation of a green fluorescent protein (GFP) reporter fused to beta-lactamases revealed that most were exported via the Sec pathway. However, the Stenotrophomonas maltophilia L2 beta-lactamase was exported via the E. coli Tat translocase, while the S. maltophilia L1 beta-lactamase was Sec dependent. These results show the possible Tat-dependent translocation of beta-lactamases in the E. coli model system. In addition, the mutation of the cytoskeleton-encoding gene mreB, which may be involved in the spatial organization of penicillin-binding proteins, decreased the MIC of beta-lactams for beta-lactamase-producing E. coli. These findings provide new knowledge about beta-lactamase translocation, a putative new target for addressing beta-lactamase-mediated resistance.