The identification of MUC1 tumor-associated Tn antigen (αGalpNAc(1->O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays (MA) and saturation transfer difference NMR (STD-NMR), we have characterized the fine-epitope mapping of a MUC1 chemi-cal library (naked and Tn-glycosylated) towards two fami-lies of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recog-nize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs showed a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing out the role of the underly-ing amino acid (Ser or Thr) in the binding process. The strategy reported can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition with particular relevance for the development of Tn-MUC1 based anticancer vaccines.