Abstract Background Mallory-Denk bodies (MDBs) are characteristic proteins and inflammatory aggregates appeared in drug-induced chronic liver injury and various chronic liver disease. MDBs formation is often accompanied with mitochondrial damage and inflammatory environment. However, how mitochondrial damage and inflammatory response affect the pathological process of MDBs is largely unknown. The scope of this study was to provide unprecedented insights into the mechanism of MDBs pathogenesis and the potential target of transforming growth factor beta (TGF-β)/JNK axis in the treatment of MDB-related chronic liver disease. Methods Single-nucleus RNA sequencing (snRNA-seq), typical MDB-related multiplex immunofluorescence staining and functional in vitro and in vivo experiments were performed to investigate the potential mechanisms of TGF-β/JNK axis in the process of MDBs formation in chronic liver injury. Furthermore, the TGF-β/JNK axis has been demonstrated to be activated in clinical patients with metabolic dysfunction-associated steatotic liver disease (MASLD), which implies the potential clinical implications of the TGF-β/JNK axis. Results Our study delineates a hepatocyte-macrophage crosstalk axis wherein TGF-β/TGF-βR dysregulation drives JNK-mediated MDBs pathogenesis. Mechanistically, the activation of JNK pathway upregulates downstream c-JUN expression (p