Colorectal cancer is the third most prevalent cancer type worldwide and contributes to a significant percentage of cancer-related mortality. Recent studies have shown that the CXCR5+CD8+ T cells present more potent proinflammatory function than CXCR5−CD8+ T cells in chronic virus infections and in follicular lymphoma, but the role of CXCR5+CD8+ T cells in colorectal cancer is yet unclear. In this study, we demonstrated that CXCR5+CD8+ T cells were very rare in peripheral blood mononuclear cells from healthy and colorectal cancer individuals, but were significantly enriched in resected tumors and tumor-associated lymph nodes. Compared to CXCR5−CD8+ T cells, the CXCR5+CD8+ T cells demonstrated significantly higher Bcl-6 expression and lower Blimp1 expression, suggesting that CXCR5+CD8+ T cells might represent a memory CD8+ T cell subset. CXCR5+CD8+ T cells also enhanced the IgG expression by autologous B cells. Under ex vivo condition, the CXCR5+CD8+ T cells demonstrated lower degranulation, TNFα expression and IFNγ expression than CXCR5−CD8+ T cells. However, after PMA + ionomycin stimulation, the degranulation and TNFα expression by CXCR5+CD8+ T cells were significantly elevated to a level comparable with CXCR5−CD8+ T cells, whereas the IFNγ expression by PMA + ionomycin-stimulated CXCR5+CD8+ T cells were significantly higher than that by CXCR5−CD8+ T cells. Following long-term TCR-stimulation, CXCR5+CD8+ T cells demonstrated significantly more potent proliferation capacity and higher IFNγ expression than CXCR5−CD8+ T cells. TCR-stimulated CXCR5+CD8+ T cells also showed a gradual downregulation in CXCR5 expression. We further found that TCR-stimulated CXCR5+CD8+ T cells demonstrated higher granzyme B production and induced more specific lysis of autologous tumor cells than CXCR5−CD8+ T cells. Together, these data demonstrate that CXCR5+CD8+ T cells represent a significant CD8+ T cell subset in colorectal tumors and have the potential to contribute to antitumor immunity, but their specific roles require further studies in vivo.