Haloarcula hispanica is one of members of the Halobacteriaceae, which displays particularly low restriction activity and is therefore important as one of the most tractable haloarchaea for archaeal genetic research.Although the Har.hispanica S-layer protein has been reported glycosylated, the S-layer glycoprotein and its glycosylation have not been investigated yet.In this study, the S-layer proteins of Har.hispanica were extracted and characterized.The S-layer was found containing two different glycoproteins which shared highly similar amino acid sequences.The genes coding for these two S-layer glycoproteins were found next to each other in the genome.Moreover,the N-and O-linked glycans were released from these two S-layer glycoproteins for structural determination.Based on the mass spectrometry and nuclear magnetic resonance, the N-glycan was determined as a branched trisaccharide containing a 225 Da residue corresponded to a 2-amino-6-sulfo-2, 6-dideoxy-quinovose, which was the first time that a naturally occurring form of sulfoquinovosamine was identified.Besides,the O-glycan was characterized as a Glcα-1, 4-Gal disaccharide by mass spectrometry combined with monosaccharide composition analysis and glycosidase treatment.The determination of the N-and O-glycan structure will be helpful for studying the diverse protein glycosylation pathways in archaea utilizing Haloarcula hispanica as a new model.