Since protein glycosylation was found in prokaryotes, multiple O-glycosylation mechanisms in the bacteria from varied genera have been reported. According to the requirement of O-oligosaccharyltransferase (OTase), the O-glycosylation in prokaryotes was divided into OTase-independent and OTase-dependent. Furthermore, the two glycosylation mechanisms were elucidated in detail. The insight into various O-glycosylation mechanisms in prokaryotes would facilitate the application of these pathways to explore targeted glycoproteins.