Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease nagana. T. brucei is dependent on glycoproteins for its survival and infectivity throughout its life cycle. Here we report the functional characterization of TbGT3, a glycosyltransferase expressed in the bloodstream and procyclic-form of the parasite. Bloodstream and procyclic-form TbGT3 conditional null mutants were created and both exhibited normal growth under permissive and non-permissive conditions. Under non-permissive conditions, the normal glycosylation of the major glycoprotein of bloodstream form T. brucei, the variant surface glycoprotein, and the absence of major alterations in lectin binding to other glycoproteins suggested that the major function of TbGT3 occurs in the procyclic form of the parasite. Consistent with this, the major surface glycoprotein of the procyclic form, procyclin, exhibited a marked reduction in molecular weight due to changes in glycosylphosphatidylinositol (GPI) anchor side-chains. Structural analysis of the mutant procyclin GPI anchors indicated that TbGT3 encodes a UDP-Gal: beta-GlcNAc-GPI beta1-3 Gal transferase. Despite the alterations in GPI anchor side chains, TbGT3 conditional null mutants remained infectious to tsetse flies under non-permissive conditions.