Arabinogalactan proteins (AGPs) are highly glycosylated proteins that play pivotal roles in diverse develop mental processes in plants. Type-II AG glycans, mostly O-linked to the hydroxyproline residues of the pro- tein backbone, account for up to 95% w/w of the AGP, but their functions are still largely unclear. Cotton fibers are extremely elongated single-cell trichomes on the seed epidermis; however, little is known of the molecular basis governing the regulation of fiber cell development. Here, we characterized the role of a CAZy glycosyltransferase 31 (GT31) family member, GhGalT1, in cotton fiber development. The fiber length of the transgenic cotton overexpressing GhGalT1 was shorter than that of the wild type, whereas in the GhGalT1-silenced lines there was a notable increase in fiber length compared with wild type. The carbohy- drate moieties of AGPs were altered in fibers of GhGalT1 transgenic cotton. The galactose: arabinose ratio of AG glycans was higher in GhGalT1 overexpression fibers, but was lower in GhGalT1-silenced lines, com- pared with that in the wild type. Overexpression of GhGalT1 upregulates transcript levels of a broad range of cell wall-related genes, especially the fasciclin-like AGP (FLA) backbone genes. An enzyme activity assay demonstrated that GhGalT1 is a b-1,3-galactosyltransferase (b-1,3-GalT) involved in biosynthesis of the b-1,3-galactan backbone of the type-II AG glycans of AGPs. We also show that GhGalT1 can form homo- and heterodimers with other cotton GT31 family members to facilitate AG glycan assembly of AGPs. Thus, our data demonstrate that GhGalT1 influences cotton fiber development via controlling the glycosylation of AGPs, especially FLAs.