Gram-negative bacteria, mitochondria and chloroplasts contain outer membrane proteins (OMPs) characterized by a transmembrane domain with a β barrel structure. Most OMPs have a diagnostic β-signal imprinted in the amino acid sequence of the final β-strand (-1 strand) of the β barrel. Molecular chaperones of the Omp85 superfamily, such as BamA in the bacterial BAM complex, recognizes the β-signal and then catalyze the folding of the OMP into its membrane-embedded β barrel structure. Here, we reconstituted OMP assembly in vitro to study this process with five model OMPs, revealing that a critical assembly signal exists in the fifth β-strand from the C-terminus (-5 strand). This signal was shown to drive a critical insertion step of the OMP assembly process but is not required for the initial recognition step between the OMP and BamA. Furthermore, we identified the receptor for this -5 signal as BamD, a second essential subunit of the BAM complex. Distinct binding sites for the β-signal and the -5 signal, were identified on BamD. Comparative sequence analysis showed that the -5 signal is a conserved feature in bacterial OMPs, and that mitochondrial OMPs also contains -5 signal motif positioned in the fifth from last strand of their β barrel structure. We propose the -5 rule as a conserved feature of the process of OMP assembly in bacteria and the organelles of eukaryotes.