We have characterized a β-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana, which is involved in the biosynthesis of type II arabinogalactan (AG). This enzyme belongs to Carbohydrate Active Enzyme Glycosyltransferase (GT) family GT14. The protein was localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. The soluble catalytic domain expressed in Pichia pastoristransferred glucuronic acid (GlcA) to β-1,6-galactooligosaccharides ranging from DP3 to 11 and β-1,3-galactooligosaccharides of DP5 and 7, indicating the enzyme is a glucuronosyltransferase modifying both β-1,6- and β-1,3-galactan present in type II AG. Two allelic T-DNA insertional mutant lines showed 20-35% enhanced cell elongation during seedling growth compared to wild type. Analyses of AG isolated from the mutants revealed a reduction of GlcA substitution on Gal-β-1,6-Gal and β-1,3-Gal, indicating anin vivo role of AtGlcAT14A in the synthesis of those structures in type II AG.Moreover, a relative increase in levels of 3-, 6-, and 3,6-linked galactose (Gal) and reduced levels of 3-, 2-, and 2,5-linked arabinose (Ara) were seen, suggesting that the mutation inAtGlcAT14Aresults in a relative increase of the longer and branched β-1,3- and β-1,6-galactan. This increase of galactosylation in the mutants is most likelycaused by increased availability of O6 position of Gal, which are shared acceptor sites for AtGlcAT14A and GalTs in the synthesis of type II AG, and thus addition of GlcA may terminate Gal chain extension. We discuss a role of the glucuronosyltransferase in the biosynthesis of type II AG with a biological role during seedling growth. This article is protected by copyright. All rights reserved.