The genome of the thermophilic bacterium, Aeribacillus pallidus 8, encodes the bacteriocin pallidocin. It belongs to the small class of glycocins and is posttranslationally modified, containing an S-linked glucose on a specific Cys residue. In this study, the pallidocin biosynthetic machinery is cloned and expressed in Escherichia coli to achieve its full biosynthesis and modification. It targets other thermophilic bacteria with potent activity, demonstrated by a low minimum inhibitory concentration (MIC) value. Moreover, the characterized biosynthetic machinery is employed to produce two other glycopeptides Hyp1 and Hyp2. Pallidocin and Hyp1 exhibit antibacterial activity against closely related thermophilic bacteria and some Bacillus sp. strains. Thus, heterologous expression of a glycocin biosynthetic gene cluster including an S-glycosyltransferase provides a good tool for production of hypothetical glycocins encoded by various bacterial genomes and allows rapid in vivo screening.