Uridine diphosphate (UDP)-GalNAc∶polypeptide N-acetylgalactosaminyltransferase (ppGalNAcT) catalyzes the initial step in mucin type O-glycosylation in the Golgi apparatus. Here generation and characterization of a polyclonal antibody to human ppGalNAcT2 were described. The subcellular location of ppGalNAcT2 in SGC7901 cell line was investigated using Western blot analysis of fractionated cell extracts and confocal microscopy with this antibody and two Golgi markers: Golgi SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) of 28 ku (GS28) and trans-Golgi network (TGN) 38, markers for the cis- and trans-Golgi apparatus, respectively. Morphometric analyses indicated that ～60% of the ppGalNAcT2 signal colocalized with the GS28, while ～36% of the cis-Golgi marker colocalized with the ppGalNAcT2. Approximately 34% of the ppGalNAcT2 signal colocalized with the TGN38, whereas 38% of the trans-Golgi marker colocalized with the ppGalNAcT2. The results provide unequivocal evidence for the location of ppGalNAcT2 within the Golgi apparatus, and further highlight the importance of this organelle in the initiation of O-linked glycosylation.