Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is observed when Tn antigen is located at Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues -Arg and Asp- present in the PDTR sequence noteworthy establish fundamental interactions with the lectin surface as well as fixing the conformation of the peptide backbone, favoring the presentation of the sugar moiety towards the lectin. These results may help to better understand the glycopeptide-lectin interactions and may contribute to engineer new binding sites, allowing the design of novel glycosensors for Tn antigen detection.