ABSTRACTGram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. InEnterobacteriaceae, the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughoutEnterobacteriaceae, the function of which is poorly understood. Previously, we identified several genes inEscherichia coliK-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these,yhdP, a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants ofyhdPwith loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability fromyhdPdeletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore,yhdPdeletion changed cellular ECA levels andyhdPwas found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability withyhdPdeletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found withyhdPdeletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP.IMPORTANCEEnterobacterial common antigen (ECA) is a surface antigen made by all members ofEnterobacteriaceae, including many clinically relevant genera (e.g.,Escherichia,Klebsiella,Yersinia). Although this surface-exposed molecule is conserved throughoutEnterobacteriaceae, very few functions have been ascribed to it. Here, we have determined that the periplasmic form of ECA, cyclic ECA, plays a role in maintaining the outer membrane permeability barrier. This activity is controlled by a protein of unknown function, YhdP, and deletion ofyhdPdamages the OM permeability barrier in a cyclic ECA-dependent manner, allowing harmful molecules such as antibiotics into the cell. This role in maintenance of the envelope permeability barrier is the first time a phenotype has been described for cyclic ECA. As the Gram-negative envelope is generally impermeable to antibiotics, understanding the mechanisms through which the barrier is maintained and antibiotics are excluded may lead to improved antibiotic delivery.