A number of proteobacteria carry the genetic information to perform N-linked glycosylation, but only the protein glycosylation (pgl) pathway of Campylobacter jejuni has been studied to date. Here, we report that the pgl gene cluster of Campylobacter lari encodes for a functional glycosylation machinery that can be reconstituted in Escherichia coli. We determined that the N-glycan produced in this system consisted of a linear hexasaccharide. We found that the oligosaccharyltransferase (OST) of C. lari conserved a predominant specificity for the primary sequence D/E-X(-1)-N-X(+1)-S/T (where X(-1) and X(+1) can be any amino acid but proline). At the same time, we observed that this enzyme exhibited a relaxed specificity toward the acceptor site and modified asparagine residues of a protein at sequences DANSG and NNNST. Moreover, C. lari pgl glycosylated a native E. coli protein. Bacterial N-glycosylation appears as a useful tool to establish a molecular description of how single-subunit OSTs perform selection of glycosyl acceptor sites.