CD1d-restricted invariant natural killer T (iNKT) cells actively patrol the liver and possess valuable antitumor potential. However, clinical trials evaluating administration of iNKT cell-specific ago-nist a-galactosylceramide (a-GalCer) have failed to achieve obvious tumor regression. Improving the efficacy of iNKT cell-based immunotherapy requires a better understanding of the factors restraining the clinical benefits. In the context of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), we found circu-lating and hepatic iNKT cells were hyperactivated but demonstrated imbalances in ratio and defective a-GalCer responsiveness. Exog-enous IL2 helped to expand residual a-GalCer-responsive clones with reduced T-cell receptor diversity. However, transcriptome-wide analysis revealed activation of the senescence-associated secretory phenotype and dampened cytotoxicity in iNKT cells, weakening their immune surveillance capacity. The senescent status of iNKT cells from the patients was further illustrated by cell-cycle arrest, impaired telomere maintenance, perturbed calcium trans-port-related biological processes, and altered metabolism. Lipidomic profiling revealed the accumulation of long-chain acylcarnitines (LCAC) and aberrant lipid metabolism in HCC tissue. Exogenous LCACs, especially palmitoyl-carnitine and stearoyl-carnitine, inhib-ited iNKT cell expansion and promoted senescence. Collectively, our results provide deeper insights into iNKT cell dysregulation and identify a cell senescence-associated challenge for iNKT cell-based immunotherapy in HBV-related HCC. The mechanistic links between iNKT cell senescence and accumulated LCACs suggest new targets for anti-HCC immunotherapies.Significance: Patients with HBV-related HCC exhibit a cell senescence-associated dysregulation of invariant natural killer cells that is related to altered lipid metabolism and accumulated LCACs in tumor tissue.