OBJECTIVE: This study investigated the role of MDK (Midkine) in hepatocellular carcinoma (HCC) through bioinformatics analysis and experimental validation, focusing on its relationship with tumor immune microenvironment and patient prognosis. METHODS: We employed the GEPIA database to analyze MDK expression patterns across cancer types and specifically in HCC versus normal tissues. MDK expression was validated through immunohistochemistry (IHC) in 100 paired HCC and adjacent tissue samples. Survival analyses were conducted using Kaplan-Meier and Cox regression methods. The relationship between MDK expression and immune cell infiltration was investigated using TIMER 2.0 database and verified through IHC staining of immune cell markers. RESULTS: MDK expression was significantly elevated in HCC tissues compared to adjacent normal tissues. High MDK expression strongly correlated with tumor number, vascular invasion, advanced clinical stage and poor prognosis, serving as an independent prognostic factor. Notably, elevated MDK expression predicted poor outcomes in patients receiving immunotherapy. Database analysis and IHC analysis revealed that MDK expression positively correlated with regulatory T (Treg) cell infiltration while negatively correlating with natural killer (NK) cell presence, suggesting its role in shaping the tumor immune microenvironment. CONCLUSION: High MDK expression in HCC correlates with unfavorable patient outcomes and impacts immune cell infiltration. MDK may serve as a novel prognostic biomarker and potential therapeutic target in HCC treatment.