T-cell-based immunotherapy holds promise for eliminating cancer through T-cell activation. However, prolonged interaction between T cells and tumors and the presence of immunosuppressive factors can diminish T-cell cytotoxicity, leading to treatment failure. Here, ribonuclease 1 (RNase1), which degrades RNA, reduced the expression of effector cytokines and increases immune checkpoint protein levels, inducing T-cell dysfunction. RNase1 expression is positively associated with exhausted T-cell gene signatures and immune checkpoint proteins across several cancer types. Cancer cells expressing RNase1 are resistant to CD8