In this study, we explored the cross-talk between phosphorylation and O-GlcNAcylation using the pancreatic ductal cell line PANC-1 as a model. Proteome and phosphoproteome changes were measured for cells treated with OSMI-1, a specific inhibitor to O-GlcNAc transferase (OGT) and Thiamet G, a specific inhibitor to O-GlcNAcase (OGA). Among the 8938 phosphorylation sites quantified, 1399 phosphosites on 870 proteins and 1480 phosphosites on 896 proteins were significantly altered by OSMI-1 and TMG treatment, respectively, demonstrating extensive cross-talk between O-GlcNAcylation and phosphorylation. In-depth analysis revealed widespread phosphorylation changes of the kinome and phosphatome, even after a short-term perturbation with inhibitors to O-GlcNAc cycling enzymes. Notably, we identified that phosphorylation of OGA at S364 is specifically mediated by casein kinase 2 alpha (CK2α), which was supported by phosphoproteomic profiling, kinase inhibition experiments, and in vitro kinase assays. These results uncover a regulatory mechanism underlying glycosylation-dependent cellular signaling through the interplay of phosphorylation and O-GlcNAcylation.