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Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate xyloglucan xylosyltransferase 1 (XXT1), a GT involved in the synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from uridine diphosphate (UDP)-xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable k cat values for reactions with UDP-xylose and UDP-glucose, while reactions with UDP-arabinose and UDP-galactose are over 10-fold slower. Using k cat / K M as a measure of efficiency, UDP-xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting that there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems. [ABSTRACT FROM AUTHOR]