A one-pot synthetic methodology for the thiolation of O-acetyl-galactoglucomannan (AcGGM) was developed to merge hemicellulose chemistry with click chemistry. This was realized by the AcGGM-mediated nucleophilic ring-opening of gamma-thiobutyrolactone via the activation of the polysaccharide pendant hydroxyl groups. The incorporation of thiol functionalities onto the hemicellulose backbone was visualized by H-1 and C-13 NMR spectroscopy and was assessed by an Ellmans reagent assay of the thiol groups. The versatility of the thiolated AcGGM was elaborated and demonstrated by conducting several postmodification reactions together with hydrogel formation utilizing thiol-ene and thiol-Michael addition click reactions. The one-pot synthesis of thiolated AcGGM is a straightforward approach that can expand the applications of hemicelluloses derived from biomass by employing click chemistry.