Protein post-translational modification (PTM) increases the functional diversity of the proteome and regulates numerous biological processes in eukaryotes. Two types of PTMs, O-linked-acetyl glucosamine modification (O-GlcNAc) and phosphorylation have been identified on the same amino acid, are considered as Yin-Yang modification for their antagonistic function recently. Vernalization, a prolonged cold exposure promoted flowering, is important for grain yield in temperate cereals, such as winter wheat. O-GlcNAcylation on TaGRP2 and phosphorylation on VER2 are involved in regulation of vernalization response (VRN) genes. However, less is known about how plant senses vernalization with general Yin-Yang modifications. Here we report that altering O-GlcNAc signaling by chemical inhibitors could change the vernalization response and affect flowering transition. Furthermore, we enriched O-GlcNAcylated and phosphorylated peptides from winter wheat plumules at different processing time points during vernalization by Lectin weak affinity chromatography (LWAC) and iTRAQ-TiO2, respectively. In total, about 200 O-GlcNAcylated proteins and 124 differential expressed phosphorylated proteins were identified by Mass Spectrum (MS). Based on GO enrichment, the identified O-GlcNAcylated proteins are mainly involved in response to abiotic stimulus and hormone, metabolic processing and gene expression. While dynamic phosphorylated proteins during vernalization participate in translation, transcription and metabolic processing. Of note, 31 proteins with both phosphorylation and O-GlcNAcylation modification were identified. Among them, TaGRP2 was further confirmed to participate in regulation of vernalization promoted flowering. The global modification profiles and genetic data at specific regulator suggested that the dynamic network of O-GlcNAcylation and phosphorylation on the key nodes regulate vernalization response and mediate flowering in wheat.