Cryptococcus neoformans is an opportunistic fungal pathogen characterized by a prominent polysaccharide capsule that envelops the cell. Although this capsule is dispensable for in vitro growth, its presence is essential for virulence. The capsule is primarily made of two xylose-containing polysaccharides, glucuronoxylomannan and galactoxylomannan. There are likely to be multiple xylosyltransferases (XTs) involved in capsule synthesis, and the activities of these enzymes are potentially important for cryptococcal virulence. A β-1,2-xylosyltransferase with specificity appropriate for capsule synthesis was purified ∼3000-fold from C. neoformans, and the corresponding gene was identified and cloned. This sequence conferred XT activity when expressed in Saccharomyces cerevisiae, which lacks endogenous XT activity. The gene, termed CXT1 for cryptococcal xylosyltransferase 1, encodes a 79-kDa type II membrane protein with an N-linked glycosylation site and two DXD motifs. These latter motifs are believed to coordinate divalent cation binding in the activity of glycosyltransferases. Site-directed mutagenesis of one DXD motif abolished Cxt1p activity, even though this activity does not depend on the addition of a divalent cation. This may indicate a novel catalytic mechanism for glycosyl transfer. Five homologs of Cxt1p were found in the genome sequence of C. neoformans and 34 within the sequences of other fungi, although none were found in other organisms. Many of the homologous proteins are similar in size to Cxt1p, and all are conserved with respect to the essential DXD motif. These proteins represent a new family of glycosyltransferases, found exclusively within the fungal kingdom.