The prevalence of the chronic metabolic disorder Type 2 diabetes mellitus (T2DM) is increasing steadily, and has even turned into an epidemic in some countries. T2DM results from defective responses to insulin and obesity is a major factor behind insulin resistance in T2DM. Insulin receptor substrate (IRS) proteins are adaptor proteins in the insulin receptor signalling pathway. The insulin signalling is controlled through tyrosine phosphorylation of IRS-1 and IRS-2, and dysregulation of IRS proteins signalling may lead to glucose intolerance and eventually insulin resistance. In this work, we suggest that both glycosylation (O-GlcNAc modification) and phosphorylation of IRS-1 and -2 are involved in the pathogenesis of T2DM. Phosphorylation and O-GlcNAc modifications (Ser1101 in IRS-1 and Ser1149 in IRS-2) proteins were determined experimentally by sandwich ELISA with specific antibodies and with bioinformatics tools. When IRS-1 (on Ser1101) and IRS-2 (Ser1149) become glycosylated following an increase in UDP-GlcNAc pools, it may contribute to insulin resistance. Whereas when the same (IRS-1 on Ser1101 and IRS-2 on Ser1149) are phosphorylated, the insulin signalling is inhibited. In this work OGlcNAc-modified proteins were specifically detected using O-Glc- NAc-specific antibodies, suggesting that elevated levels of O-GlcNAc-modified proteins are found, independently of their possible involvement in Advanced Glycation End products (AGEs). This study suggests a mechanism, which is controlled by posttranslational modifications, and may contribute to the pathogenesis of type II diabetes.