Mono-O-glycosylation of eukaryotic target proteins is the molecular mechanism of bacterial protein toxins of the family of large clostridial cytotoxins. This toxin family encompasses several high molecular mass proteins (>250 kDa) of various Clostridia species that are implicated in severe human diseases. Toxin A and toxin B from Clostridium difficile are the causative agents of pseudomembranous colitis and antibiotic-associated diarrhea. Lethal toxin and hemorrhagic toxin from Clostridium sordellii as well as alpha-toxin from Clostridium novyi are involved in the gas gangrene syndrome. The common mode of action of large clostridial cytotoxins is elicited by specific glycosylation of small GTP-binding proteins in the cytosol of target cells using activated nucleotide sugars as cosubstrates. Specific modification at a single threonine residue in the small GTPases renders these important key players of various signaling pathways inactive. This minireview intends to give an overview on structure-function analysis and mode of action of the large clostridial cytotoxins, as well as on the resulting functional consequences of glycosylation of target proteins.