Tau protein is a key player in Alzheimer’s disease as the main constituent of intracellular neurofibrillary tangles.1 Tau protein is modified by multiple post-translational modifications (PTMs) and these PTMs can affect each other in what is generally termed “PTM crosstalk”. 2 Among several post-translational modifications of Tau, both phosphorylation and O-GlcNAcylation are known to occur on at least 12 Ser/Thr sites in particular O-GlcNAcylation is well-studied in the C-terminal region of the Tau (residues 392-411 from the longest isoform of Tau; PHF-1 epitope).3O-GlcNAcylation has been involved in the modulation of tau phosphorylation levels and inhibition of tau aggregation properties while a decrease of O-GlcNAcylation could be involved in tau hyperphosphorylation.4 However, the molecular mechanisms at the basis of these observations is ill-defined. Our study aims to decipher the role of O-GlcNAcylation in the regulation of tau phosphorylation and conformation and thereby modulate aggregation. We used advanced molecular dynamics (MD) simulations in conjunction with high-resolution NMR to describe the direct O-GlcNAcylation/phosphorylation crosstalk around the PHF-1 epitope and investigate their effect on peptide conformation. Our results from the all-atom explicit solvent MD simulations that the PHF-1 peptide in the absence of post-translational modifications forms transient helix from residues Ser404-Gln410, in particular, the hydrogen bond between backbone atoms of Leu408-Ser404 and Ser404-Val399 was found to be present in these helical conformations. Our advanced MD simulations around the PHF-1 phospho-epitope show that phosphorylation and O-GlcNAcylation both disrupt the turn-like conformation. The structural changes correlated well with the NMR studies on these Tau fragments. Our findings refute the general norm in the field that phosphorylation and O-GlcNAcylation of Tau are reciprocally antagonistic.