Mucin type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Deficiencies of individual GalNAc-Ts cause subtle distinct phenotypes in insect and mu-rine models, and altered expression patterns of GalNAc-Ts have been identified as prognostic cancer markers. Yet, we have little understanding of the cell and tissue specific functions of the individual isoforms. We have recently used precise genetic engineering to target the human C1GalT1 chaperone COSMC to generate stable cells and tissue models with homogenous truncated GalNAc O-glycans, demonstrating a malignant phenotype. We now extend these studies to characterize the impact of site-specific glycosylation on epithe-lial differentiation and malignant transformation. Using an organotypic model system equipped with cells with and without GalNAc-T1,-T2, or-T3, we identify distinct pheno-types, and selective effects on specific cellular pathways identified via differential transcriptomic, phosphoproteomic, and proteomic analyses. In addition, we find non-redundant O-glycosylation performed by single isoforms using quantitative differential O-glycoproteomics and identify isolated sites on proteins involved in cell adhesion, differentiation, and stress response. Importantly, knock-out of GalNAc-T1,-T2, or-T3 does not lead to malignant transformation, in contrast to homogeneous truncation of O-glycans. While GalNAc-T1-T3 are abundantly expressed in most healthy tissues, we found that GalNAc-T6 was highly upregulated in colon adeno-carcinomas but absent in normal-appearing adjacent tissue samples, suggesting that it plays a role in colon carcinogenesis. GalNAc-T6 expression was associated with a cancer-like growth pattern, whereas GalNAc-T6 knockout cells showed more normal differentiation patterns, normalized cell-cell adhesion, and formed crypts in tissue cultures. In addition, several GalNAc-T6 specific targets were identified by O-glycoproteomic analysis. Taken together, these data strongly suggest that individual GalNAc-T isoforms glycosylate a subset of specific targets that play important roles in tissue differ-entiation, homeostasis and oncogenesis.