Membrane vesicle (MV) release remains undefined, despite its conservation among replicating Gram‐negative bacteria both in vitro and in vivo. Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane protein–peptidoglycan (OM–PG) and OM protein–inner membrane protein (OM–PG–IM) interactions within the envelope structure. Modulation of OM–PG and OM–PG–IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co‐ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM–PG and OM–PG–IM associations within the envelope structure, thus releasing OM as MVs.