Cell surface glycosylation is an important element in defining the life of pathogenic bacteria. Tannerella forsythia is a Gram-negative, anaerobic periodontal pathogen inhabiting the subgingival plaque biofilms. It is completely covered by a two-dimensional crystalline surface layer (S-layer) composed of two glycoproteins. Although the S-layer has previously been shown to delay the bacterium's recognition by the innate immune system, we characterize here the S-layer protein O-glycosylation as a potential virulence factor. The T. forsythia S-layer glycan was elucidated by a combination of electrospray ionization-tandem mass spectrometry and nuclear magnetic resonance spectroscopy as an oligosaccharide with the structure 4-Me-β-ManpNAcCONH 2-(1→3)- [Pse5Am7Gc-(2→4)-]-β-ManpNAcA-(1→4)-[4- Me-α-Galp- (1→2)-]-α-Fucp-(1→4)-[-α-Xylp-(1→3)- ]-β-GlcpA-(1→3)- [-β-Digp-(1→2)-]-α-Galp, which is O-glycosidically linked to distinct serine and threonine residues within the threeamino acid motif (D)(S/T)(A/I/L/M/T/V) on either S-layer protein. This S-layer glycan obviously impacts the life style of T. forsythia because increased biofilm formation of an UDP-N-acetylmannosaminuronic acid dehydrogenase mutant can be correlated with the presence of truncated S-layer glycans. We found that several other proteins of T. forsythia are modified with that specific oligosaccharide. Proteomics identified two of them as being among previously classified antigenic outer membrane proteins that are up-regulated under biofilm conditions, in addition to two predicted antigenic lipoproteins. Theoretical analysis of the S-layer O-glycosylation of T. forsythia indicates the involvement of a 6.8-kb gene locus that is conserved among different bacteria from the Bacteroidetes phylum. Together, these findings reveal the presence of a protein O-glycosylation system in T. forsythia that is essential for creating a rich glycoproteome pinpointing a possible relevance for the virulence of this bacterium.