Outer membrane vesicles (OMVs) act as carriers of bacterial products such as plasmids and resistance determinants, including metallo-beta-lactamases. The lipidated, membrane-anchored metallo-beta-lactamase NDM-1 can be detected in Gram-negative OMVs. The soluble domain of NDM-1 also forms electrostatic interactions with the membrane. Here, we show that these interactions promote its packaging into OMVs produced by Escherichia coli. We report that favorable electrostatic protein-membrane interactions are also at work in the soluble enzyme IMP-1 while being absent in VIM-2. These interactions correlate with an enhanced incorporation of IMP-1 compared to VIM-2 into OMVs. Disruption of these interactions in NDM-1 and IMP-1 impairs their inclusion into vesicles, confirming their role in defining the protein cargo in OMVs. These results also indicate that packaging of metallo-beta-lactamases into vesicles in their active form is a common phenomenon that involves cargo selection based on specific molecular interactions.