Updated project metadata. Mass spectrometry and NMR were used to characterize the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. The S-layer protein was identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690 and is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide alpha-GlcNAc-4-beta-GlcNAc3NGaAN-4-beta-Glc-Asn where the 2nd residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide beta-GlcNAc3NAcAN-4-beta-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.