<p>The DnaJ/Hsp40 family member B6 (DNAJB6) is a protein aggregation inhibitor, and its dysregulation has been associated with various diseases. Although early studies implicated DNAJB6’s role in tumor progression, its prognostic and immunotherapy predictive value across cancers remain undefined. Integrated multi-omics data analysis revealed significant upregulation of DNAJB6 in multiple tumors, particularly in liver hepatocellular carcinoma (LIHC), supported by immunohistochemistry of liver tumor tissues and Western blot assays in HepG2 cells, and poor prognosis in LIHC patients was linked to increased DNAJB6 expression. ROC and univariate Cox analyses established DNAJB6 as an independent prognostic factor in LIHC, with good predictive value. Enrichment, immune scoring and single-cell sequencing analyses indicated that DNAJB6 was linked to immune responses, especially with myeloid-derived suppressor cell (MDSC) and exhausted CD8⁺ T cell (T_ex). Cell experiments demonstrated that siRNA targeting DNAJB6 inhibited HepG2 cells from proliferating and migrating while inducing apoptosis. Furthermore, we predicted and experimentally confirmed that DNAJB6 silencing conferred gefitinib resistance in HepG2 cells. In conclusion, our findings highlighted the significance of DNAJB6 expression in determining LIHC prognosis and immunotherapy response, as well as its potential in developing novel anti-cancer drugs and enhancing immunotherapy.</p>