Liver injury causes “transdifferentiation” of quiescent hepatic stellate cells (Q-HSCs) into wound repairing myofibroblasts (MF-HSCs), whose uncontrolled activation leads ultimately to liver fibrosis. Although this process is triggered by deep metabolic and transcriptional reprogramming, functional links between these two key events are not yet understood. Here, using a compendium of in vitro, ex vivo and in vivo models of fibrogenic liver injuries in addition to human liver samples, we report that O-linked β D N acetylglucosaminylation (O GlcNAcylation), a post-translational modification (PTM) considered as nutritional sensor, is increased during myofibroblastic activation of HSCs and is required for their pro-fibrotic activities. Mechanistically, a multi omics approach combining proteomic, epigenomic and transcriptomic data mining revealed that O GlcNAcylation controls the transcriptional program of MF-HSCs by targeting the key fibrogenic transcription factors Basonuclin 2 (BNC2) and TEA domain transcription factor 4 (TEAD4) together with the Yes associated protein 1 (YAP1) co-activator. Indeed, inhibition of protein O GlcNAcylation impedes their stability leading to decreased functionality of the BNC2/TEAD4/YAP1 complex towards promoting activation of fibrogenic transcriptional regulatory elements. Altogether, this study unravels the fibrogenic role of protein O-GlcNAcylation identifying a vulnerable regulatory process in activated HSCs.