Mucins are highly glycosylated proteins whose structural complexity and dynamic behavior are crucial for their biological function and pathological role. Understanding their conformational properties, both in free form and when interacting with mucin-binding proteins, is essential for the development of therapeutic strategies, especially in cancer immunotherapy. Nuclear magnetic resonance (NMR) spectroscopy has become a central technique for studying the conformational dynamics of mucins and mucin-like glycopeptides. In combination with molecular dynamics simulations, NMR provides detailed insights into the flexible structural ensembles that characterize these molecules in solution. This chapter provides an overview of how NMR-based approaches together with computational methods have revealed the key role of O-glycosylation in shaping the three-dimensional organization of mucins. Particular attention is given to the conformational differences induced by glycosylation at serine versus threonine residues, the effects of water-mediated interactions, and the consequences for molecular recognition by antibodies, lectins, and transferases. The structural features of mucins described in this chapter could play a key role in the development of synthetic antigens for designing cancer vaccines and drugs targeting mucin-related diseases.