The plant UDP-glycosyltransferases (UGTs) regulate several metabolic processes during root growth and development by conjugating sugar moieties to various small molecules. RsUGT71B5 is a novel UDP-glycosyltransferase in Raphanus sativus L., but its biological function is not well established. In this study, we generated RsUGT71B5-overexpressing transgenic Arabidopsis lines to determine the mechanisms by which RsUGT71B5 regulated root growth and development. Ectopic overexpression of RsUGT71B5 significantly enhanced root growth and seedling development. In culture medium supplemented with 1-3% exogenous sucrose, RsUGT71B5 overexpression increased the root length and surface area in the transgenic Arabidopsis lines compared with the wild type. Furthermore, transgenic RsUGT71B5 overexpression partially suppressed the inhibitory effects of 12% sucrose on root growth and development. RNA sequencing data analysis identified 102 differential expressed genes (DEGs), including 56 upregulated and 46 downregulated genes, in the transgenic RsUGT71B5 overexpression lines (OE). QRT-PCR analyses confirmed significant upregulation of glutathione S-transferases such as AT1G02930 (GSTF6) and AT1G02920 (GSTF7) in the transgenic RsUGT71B5 overexpression lines. KEGG pathway analyses of the DEGs showed that RsUGT71B5 overexpression regulated glutathione and sugar metabolism. In summary, this study demonstrated that RsUGT71B5 regulated root growth and development by modulating glutathione and sugar metabolism.