Bacteria secrete extracellular membrane vesicles (EMVs). Physiological functions and biotechnological applications of these lipid nanoparticles have been attracting significant attention. However, the details of the molecular basis of EMV biogenesis have not yet been fully elucidated. In our previous work, an N-terminus-substituted FAAV peptide labeled with nitrobenzoxadiazole (NBD; nFAAV5-NBD) was developed. This peptide can sense the curvature of a lipid bilayer and selectively bind to EMVs even in the presence of cells. Here, we applied nFAAV5-NBD to a genome-wide screening of hyper- and hypo-vesiculation transposon mutants of a Gram-negative bacterium, HM13, to identify the genes involved in EMV production. We analyzed the transposon insertion sites in hyper- and hypo-vesiculation mutants and identified 16 and six genes, respectively, with a transposon inserted within or near them. Targeted gene-disrupted mutants of the identified genes showed that the lack of putative dipeptidyl carboxypeptidase, glutamate synthase β-subunit, LapG protease, metallohydrolase, RNA polymerase sigma-54 factor, inactive transglutaminase, PepSY domain-containing protein, and Rhs-family protein caused EMV overproduction. On the other hand, disruption of the genes encoding putative phosphoenolpyruvate synthase, d-hexose-6-phosphate epimerase, NAD-specific glutamate dehydrogenase, and sensory box histidine kinase/response regulator decreased EMV production. This study demonstrates the utility of a novel screening method using a curvature-sensing peptide for mutants with altered EMV productivity and provides information on the genes related to EMV production.IMPORTANCEConventional methods for isolation and quantification of extracellular membrane vesicles (EMVs) are generally time-consuming. nFAAV5-NBD can detect EMVs in the culture without separating EMVs from cells. detection of EMVs using this peptide facilitated screening of the genes related to EMV production. We succeeded in identifying various genes associated with EMV production of HM13, which would contribute to the elucidation of bacterial EMV formation mechanisms. Additionally, the hyper-vesiculating mutants obtained in this study would be valuable for EMV applications, such as secreting useful substances as EMV cargoes and producing artificially functionalized EMVs.