Glycocin F (GccF), a ribosomally synthesised post-translationally modified peptide secreted by Lactobacillus plantarum KW30, rapidly inhibits the growth of susceptible bacteria at nanomolar concentrations. The NMR structure showed that GccF contains two nested disulfide bonds and two N-acetylglucosamine (GlcNAc) moieties, one β-O-linked to Ser18, and the other β-S-linked to Cys43. Glycosylated cysteine residues remain relatively rare in prokaryotes and have only been confirmed in seven other bacteriocins. Previous studies have highlighted structural features important for its activity and shown the absolute requirement for the Ser18O-linked GlcNAc. Here we show that an ostensibly very small chemical modification to this Ser, substitution of the Cα proton with a methyl group (GccFα-methylSer18), reduces the antimicrobial activity of GccF 1000-fold. A comparison of the GccFα-methylSer18 NMR structure with that of the native protein showed a marked difference in the orientation and mobility of the loop bearing Ser18 in GccFα-methylSer18, leading to markedly different positioning of its GlcNAc, suggesting that both dynamics and stereochemistry of the presentation of the GlcNAc may play an important role in the interaction of GccF with a second yet unknown but essential physiological partner.