The worldwide dissemination of metallo-β-lactamases (MBLs)， mediating resistance to carbapenem antibiotics， is a major public health problem. The extent of dissemination of MBLs such as VIM-2， SPM-1 and NDM among Gram-negative pathogens cannot be explained solely based on the associated mobile genetic elements or the resistance phenotype. Here， we report that MBL host range is determined by the impact of MBL expression on bacterial fitness. The signal peptide sequence of MBLs dictates their adaptability to each host. In uncommon hosts， inefficient processing of MBLs leads to accumulation of toxic intermediates that compromises bacterial growth. This fitness cost explains the exclusion of VIM-2 and SPM-1 from Escherichia coli and Acinetobacter baumannii， and their confinement to Pseudomonas aeruginosa. By contrast， NDMs are expressed without any apparent fitness cost in different bacteria， and are secreted into outer membrane vesicles. We propose that the successful dissemination and adaptation of MBLs to different bacterial hosts depend on protein determinants that enable host adaptability and carbapenem resistance.