In an effort to devise a safer and more effective vaccine delivery system, outer membrane vesicles (OMVs) were engineered to have properties of intrinsically low endotoxicity sufficient for the delivery of foreign antigens. Our strategy involved mutational inactivation of the MsbB (LpxM) lipid A acyltransferase to generate OMVs of reduced endotoxicity from Escherichia coli (E. coli) O157:H7. The chromosomal tagging of a foreign FLAG epitope within an OmpA-fused protein was exploited to localize the FLAG epitope in the OMVs produced by the E. coli mutant having the defined msbB and the ompA::FLAG mutations. It was confirmed that the desired fusion protein (OmpA::FLAG) was expressed and destined to the outer membrane (OM) of the E. coli mutant from which the OMVs carrying OmpA::FLAG are released during growth. A luminal localization of the FLAG epitope within the OMVs was inferred from its differential immunoprecipitation and resistance to proteolytic degradation. Thus, by using genetic engineering-based approaches, the native OMVs were modified to have both intrinsically low endotoxicity and a foreign epitope tag to establish a platform technology for development of multifunctional vaccine delivery vehicles.