Phosphorylation and O-GlcNAcylation of keratin 18 (K18) are highly dynamic and involve primarily independent K18 populations. We used in vitro phosphorylation and O-GlcNAcylation of wild-type, phospho-Ser52, glyco-Ser48, and Ser-to-Ala mutant 17mer peptides (K18 amino acids 40-56), which include the major K18 glycosylation (Ser48) and phosphorylation (Ser52) sites, to address whether each modification blocks the other. The glyco-K18 peptide blocks Ser52 phosphorylation by protein kinase C, an in vivo K18 kinase, while the phospho-K18 peptide blocks its O-GlcNAcylation. Our findings support the reciprocity of these two post-translational modifications. Therefore, regulation of protein Ser/Thr phosphorylation and glycosylation at proximal sites can be interdependent and provides a potential mechanism of counter regulation.