The follicular CXCR5+CD8+ T cells have recently emerged as a critical cell type in mediating peripheral tolerance as well as antiviral immune responses during chronic infections. In this study, we investigated the function of CXCR5+CD8+ T cells in HBV-related hepatocellular carcinoma patients. Compared to CXCR5−CD8+ T cells, CXCR5+CD8+ T cells presented elevated PD-1 expression but reduced Tim-3 and CTLA-4 expression. Upon anti-CD3/CD28 stimulation, CXCR5+CD8+ T cells demonstrated higher proliferation potency than CXCR5−CD8+ T cells, especially after PD-1 blockade. CXCR5+CD8+ T cells also demonstrated significantly higher granzyme B synthesis and release, as well as higher level of degranulation. Tumor cells were more readily eliminated by CXCR5+CD8+ T cells than by CXCR5−CD8+ T cells. Interestingly, we found that B cells were more resistant to CXCR5+CD8+ T cell-mediated killing than tumor cells, possibly through IL-10-mediated protection. In addition, the CXCR5+CD8+ T cell-mediated cytotoxic effects on tumor cells could be significantly enhanced by PD-L1 blockade. Together, we presented that in patients with in HBV-related hepatocellular carcinoma, CXCR5+CD8+ T cells could mediate tumor cell death more potently than the CXCR5−CD8+ T cells in vitro while the autologous B cells were protected.