Glycosyltransferases (GTs) are a large family of enzymes that specifically transfer sugar moieties to a diverse range of substrates. The process of bacterial glycosylation (such as biosynthesis of glycolipids, glycoproteins, and polysaccharides) has been studied extensively, yet the majority of GTs involved remains poorly characterized. Besides predicting enzymatic parameters of GTs, the resolution of three-dimensional structures of GTs can help to determine activity, donor sugar binding, and acceptor substrate binding sites. It also facilitates amino acid sequence-based structural modeling and biochemical characterization of their homologues. Here we describe a general procedure to accomplish expression and purification of soluble and active recombinant GTs. Enzymatic characterization, and crystallization of GTs, and data refinement for structural analysis are also covered in this protocol.