Significance Protein secretion is an essential determinant of bacterial physiology and virulence. Members of the Corynebacteriales order have evolved a complex cell envelope containing two membranes, a plasma membrane and an outer membrane, called the mycomembrane, which harbors mycolic acids and outer membrane proteins (OMPs) of unusual structure. Here, we have investigated the biogenesis of OMPs in Corynebacterium glutamicum and deciphered the role of O -mycoloylation in targeting OMPs to the mycomembrane. Partially enabled by our methodology, we found that the posttranslational state of major OMPs determined their presence in the outer membrane vs. the extracellular medium. We have also uncovered a short linear amino acid motif for O -acylation of proteins that seems to be preserved throughout the kingdoms.