O-GlcNAcylation on serine and threonine side chains of nuclear and cytoplasmic proteins is dynamically regulated in response to various environmental and biological stimuli. O-GlcNAcylation is remarkably similar to O-phosphorylation and appears to have a dynamic interplay with O-phosphate in cellular regulation. A systematic glyco-proteomic analysis of the affects of inhibiting specific kinases on O-GlcNAcylation should help reveal both the global and specific dynamic relationships between these two abundant post-translational modifications. Here, we report the O-GlcNAc perturbations in response to inhibition of glycogen synthase kinase-3 (GSK-3), a pivotal kinase involved in many signaling pathways. By combining immunoaffinity chromatography and SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative mass spectrometry, we identified 45 potentially O-GlcNAcylated proteins. Quantitative measurements indicated that at least 10 proteins had apparently increased O-GlcNAcylation upon GSK-3 inhibition by lithium, while, surprisingly 19 other proteins showed decreases. O-GlcNAcylation changes on a subset of the proteins were confirmed by follow-up experiments. By combining a new O-GlcNAc peptide enrichment method and -elimination followed by Michael addition with DTT (BEMAD), we also mapped the O-GlcNAc site (Ser-55) of vimentin, which showed apparently increased O-GlcNAcylation upon GSK-3 inhibition. Based on the MS data, we further investigated potential roles of O-GlcNAc on host cell factor (HCF-1), a transcription co-activator, and showed that dynamic regulation of O-GlcNAcylation on HCF-1 influenced its subcellular distribution. Taken together, these data indicated the complex interplay between phosphorylation and O-GlcNAcylation that occurs within signaling networks.