Introduction: Cancer-related fatigue is a debilitating symptom among hepatocellular carcinoma (HCC) patients, significantly impacting their quality of life. Emerging evidence suggests that Vitamin D may help alleviate fatigue; however, its causal role remains unclear.

Objective: This study aims to investigate the causal relationship between HCC, Vitamin D, and fatigue by assessing the mediating role of Vitamin D using Mendelian randomization (MR) analysis.

Methods: A two-sample MR analysis was conducted using genome-wide association study (GWAS) data. Genetic instruments for “HCC,” “Vitamin D,” and “fatigue” were obtained from the Integrative Epidemiology Unit OpenGWAS database. Inverse variance weighting, MR-Egger regression, weighted median, simple mode, and weighted mode methods, as well as sensitivity analyses such as the MR-Egger intercept, Cochran’s Q test, leave-one-out analysis, and MR Pleiotropy RESidual Sum and Outlier, were employed to ensure robustness. Finally, a two-step MR analysis was performed to quantify Vitamin D’s mediation effect.

Results: HCC showed a significant causal effect on increased fatigue risk (odds ratio = 1.78; 95% confidence interval: 1.16–2.74; p<0.05) and decreased Vitamin D levels (β = −12.61; p<0.05). Higher Vitamin D levels were associated with reduced fatigue severity (β = −0.01; p<0.05). Mediation analysis indicated that 21.74% of the effect of HCC on fatigue was mediated by Vitamin D. Sensitivity analyses confirmed the robustness of these findings.

Conclusion: Vitamin D may partially mediate the HCC–fatigue relationship inferred from genetic proxies for chronic fatigue syndrome, highlighting its potential, albeit preliminary, role as a therapeutic target for cancer-related fatigue management. Future clinical trials should evaluate the efficacy of Vitamin D supplementation in alleviating fatigue among HCC patients.