CD8⁺ T cells play critical role in controlling the metastasis and prognosis of cancer. Controversy remains as to the contribution of Notch signaling pathway in modulation of CD8⁺ T cells activity and development of tumorigenesis. Thus, the aim of the current study was to investigate the immunoregulatory role of Notch signaling pathway to peripheral and tumor-infiltrating CD8⁺ T cells in patients with colorectal carcinoma. A total of 46 patients with colorectal carcinoma and 20 health individuals were enrolled, and CD8⁺ T cells were purified from both peripheral bloods and carcinoma specimens. Cytolytic and noncytolytic functions of CD8⁺ T cells in response to Notch signaling inhibition were evaluated by measurements of lactate dehydrogenase release and proinflammatory cytokines production in both direct and indirect contact co-culture system to target HT29 cells. Cellular proliferation and inhibitory receptors expression in CD8⁺ T cells were also assessed by CCK-8 method and flow cytometry. There was no remarkable difference in percentage of CD8⁺ T cells between healthy individuals and patients with colorectal carcinoma. Notch1/2 and Hes1/5 mRNAs were elevated expressed in tumor-infiltrating CD8⁺ T cells in patients with colorectal carcinoma, however, did not correlated with tumor differentiation or stages. CD8⁺ T cells from healthy individuals presented stronger cytotoxicity, which was not affected by Notch signaling inhibitor. Inhibition of Notch signaling pathway not only promoted cytotoxicity of tumor-infiltrating CD8⁺ T cells, but also enhanced proinflammatory cytokines (including IFN-γ, TNF-α, IL-1β, IL-6, and IL-8) production by CD8⁺ T cells from patients with colorectal carcinoma. This process was accompanied by decreased expression of PD-1 in CD8⁺ T cells without influencing cellular proliferation. Our results indicated a potential immunosuppressive property of Notch signaling pathway, which dampened both cytolytic and noncytolytic functions of CD8⁺ T cells probably via induction of PD-1 in patients with colorectal carcinoma.