Current chemotherapeutic efficacy is limited by the rapid development of multidrug resistance (MDR) in hepatocellular carcinoma (HCC). In this study, 66 MDR-related hub genes in drug-resistant HCC were identified through combined analysis of differential expressed genes (DEGs), gene functional enrichment, Cox proportional regression, weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network construction. A prognostic risk model was established through the LASSO-Cox regression analysis. Based on the comparison of gene mutation frequency, tumor mutation burden (TMB) and immune infiltration in high- and low-risk groups, we explored the relationships between the MDR-related hub genes and immune regulation. The competitive endogenous RNA (ceRNA) network and associated non-coding RNAs (ncRNAs) were predicted to investigate the potential mechanisms. Five MDR-related hub genes in drug-resistant HCC were finally confirmed, namely ABCB6, FLNC, MCC, NAV3 and TRIM9. TRIM9 was identified as the most significant gene enhancing MDR. Inhibiting TRIM9 caused a decrease in the IC