Gram-negative bacteria produce outer membrane vesicles (OMVs), which contain biologically active proteins and perform diverse biological functions. Unlike other secretion mechanisms, OMVs enable the secretion of insoluble molecules alongside soluble material. Key characteristics of OMV biogenesis include outward bulging of areas lacking membrane-peptidoglycan bonds, upregulation of vesicle production without losing outer membrane integrity, enrichment or exclusion of certain proteins and lipids, and membrane fission without direct energy from ATP/GTP hydrolysis. OMVs play roles in bacterial survival, nutrient acquisition, biofilm development, and pathogenesis. The production of OMVs is a stress response and is regulated by various conditions and genes. OMVs can act as delivery vehicles, nucleators in bacterial communities, and contributors to bacterial survival and virulence. They can also facilitate horizontal gene transfer and endocytosis into eukaryotic cells. OMVs are regulated by growth conditions and genetic factors, and their content is characterized using mass spectrometry. The biogenesis of OMVs involves outward bulging, packaging of specific components, and membrane fission, likely driven by non-exclusive events depending on physiological circumstances or species.Gram-negative bacteria produce outer membrane vesicles (OMVs), which contain biologically active proteins and perform diverse biological functions. Unlike other secretion mechanisms, OMVs enable the secretion of insoluble molecules alongside soluble material. Key characteristics of OMV biogenesis include outward bulging of areas lacking membrane-peptidoglycan bonds, upregulation of vesicle production without losing outer membrane integrity, enrichment or exclusion of certain proteins and lipids, and membrane fission without direct energy from ATP/GTP hydrolysis. OMVs play roles in bacterial survival, nutrient acquisition, biofilm development, and pathogenesis. The production of OMVs is a stress response and is regulated by various conditions and genes. OMVs can act as delivery vehicles, nucleators in bacterial communities, and contributors to bacterial survival and virulence. They can also facilitate horizontal gene transfer and endocytosis into eukaryotic cells. OMVs are regulated by growth conditions and genetic factors, and their content is characterized using mass spectrometry. The biogenesis of OMVs involves outward bulging, packaging of specific components, and membrane fission, likely driven by non-exclusive events depending on physiological circumstances or species.