Heparan and chondroitin sulfates play essential roles in growth factor signaling during development and share a common linkage tetrasaccharide structure, GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser. In the present study, we identified the Drosophila proteoglycan UDP-galactose:β-xylose β1,4-galactosyltransferase I (dβ4GalTI), and determined its substrate specificity. The enzyme transferred a Gal to the -β-xylose (Xyl) residue, confirming it to be the Drosophila ortholog of human proteoglycan UDP-galactose:β-xylose β1,4-galactosyltransferase I. Then we established UAS-dβ4GalTI-IR fly lines containing an inverted repeat of dβ4GalTI ligated to the upstream activating sequence (UAS) promoter, a target of GAL4, and observed the F1 generation of the cross between the UAS-dβ4GalTI-IR fly and the Act5C-GAL4 fly. In the F1, double-stranded RNA of dβ4GalTI is expressed ubiquitously under the control of a cytoplasmic actin promoter to induce the silencing of the dβ4GalTI gene. The expression of the target gene was disrupted specifically, and the degree of interference was correlated with phenotype. The lethality among the progeny proved that β4GalTI is essential for viability. This study is the first to use reverse genetics, RNA interference, to study the Drosophila glycosyltransferase systematically.