We report the functional characterization of a new UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) (EC 188.8.131.52) from the human disease-causing parasite, Toxoplasma gondii. This glycosyltransferase is denoted as T. gondii ppGalNAc-T3. These enzymes are responsible for the initial step of mucin-type O-glycosylation: the transfer of GalNAc from the UDP-GalNAc nucleotide sugar donor onto a peptide acceptor. Following an in silico analysis of the publicly available T. gondii DNA database, we used molecular biology approaches to identify and isolate the cDNA encoding this enzyme. The resulting type II membrane protein contains N-terminal cytoplasmic, transmembrane, and C-terminal lumenal domains. Conceptual translation of the cDNA sequence also reveals a stem region and the presence of several important sequence motifs. When the recombinant construct was expressed in stably transfected Drosophila melanogaster S2 cells, the purified protein exhibited glycosyltransferase activity in vitro against glycopeptide, but not "naked" peptide, acceptors. In addition, using reverse transcriptase-PCR, T. gondii ppGalNAc-T3 mRNA was equivalently expressed during the tachyzoite and bradyzoite developmental stages. The identification of T. gondii ppGalNAc-T3 as a functional "follow-up" glycopeptide glycosyltransferase further confirms that this human parasite has its own enzymatic O-glycosylation machinery.