The six-component maintenance of lipid asymmetry (Mla) system is responsible for retrograde transport of phospholipids, ensuring the barrier function of the Gram-negative cell envelope. Located within the outer membrane MlaA (VacJ) likely acts as a channel to shuttle phospholipids from the outer leaflet, bypassing the inner leaflet. In Neisseria gonorrhoeae, we previously discovered MlaA, encoded by the ngo2121 genetic locus, as a proteome-derived new vaccine and therapeutic target against this serious public health threat. Our follow-up with phenotype microarrays linked the loss of MlaA with an extensive chemical sensitivity phenome. For the current study, we investigated the role of gonococcal MlaA in bacterial physiology and pathogenicity. Quantitative proteomics were applied to cell envelopes and membrane vesicles derived from wild type and ∆mlaA bacteria to examine the alterations in protein composition induced by the loss of MlaA from the outer membrane, as well as to determine whether the abundance of known or potential virulence factors was altered in the mutant. We determined that membrane vesicles were more highly affected by the loss of MlaA than were cell envelopes, and that both cell envelopes and membrane vesicles derived from the ∆mlaA mutant were enriched with adhesins and other virulence factors.