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Background Cytokinins are one of the five major hormones families in plants and are important for their normal growth and environmental adaptability. In plants, cytokinins are mostly present as glycosides in plants, and their glycosylation modifications are catalyzed by family 1 glycosyltransferases. Current research on cytokinin glycosylation has focused on the biochemical identification of enzymes and the analysis of metabolites in Arabidopsis. There are few studies that examine how cytokinin glycosylation affects its synthesis and accumulation in plants. It is particularly important to understand these processes in food crops such as rice ( Oryza sativa ); however, to date, cytokinin glycosyltransferase genes in rice have not been reported. Results In this study, we identified eight rice genes that were functionally homologous to an Arabidopsis cytokinin glycosyltransferase gene. These genes were cloned and expressed in a prokaryotic system to obtain their purified proteins. Through enzymatic analysis and liquid chromatography-mass spectrometry, a single rice glycosyltransferase, Os6 , was identified that glycosylated cytokinin in vitro. Os6 was overexpressed in Arabidopsis, and the extraction of cytokinin glycosides showed that Os6 is functionally active in planta . Conclusions The identification and characterization of the first cytokinin glycosyltransferase from rice is important for future studies on the cytokinin metabolic pathway in rice. An improved understanding of rice cytokinin glycosyltransferases may facilitate genetic improvements in rice quality. Electronic supplementary material The online version of this article (10.1186/s12284-019-0279-9) contains supplementary material, which is available to authorized users.