O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) regulates many cellular processes including the cell cycle, cell signaling, and protein trafficking. Dysregulation of O-GlcNAcylation may be involved in the development of insulin resistance and type 2 diabetes. Therefore, it is necessary to identify cellular proteins that are induced by elevated O-GlcNAcylation. Here, using adenosine 5'-triphosphate affinity chromatography, we employed a proteomic approach in order to identify differentially expressed proteins in response to treatment with the O-GlcNAcase inhibitor, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), in mouse C2C12 myotube cells. Among 205 selected genes, we identified 68 nucleotide-binding proteins, 14 proteins that have adenosinetriphosphatase activity, and 10 proteins with ligase activity. Upregulation of proteins, including ubiquitin-activating enzyme E1, proteasome subunit 20S, cullin-associated NEDD8-dissociated protein 1, ezrin, and downregulation of the protein nucleoside diphosphate kinase B, were confirmed by western blot analysis. In particular, we found that the protein ubiquitination level in C2C12 cells was increased by PUGNAc treatment. This is the first report of quantitative proteomic profiles of myotube cells after treatment with PUGNAc, and our results demonstrate the potential to enhance understanding of the relationship between insulin resistance, O-GlcNAc, and PUGNAc in the future.