<p>5-Fluorouracil (5-FU) is the cornerstone of chemotherapy for patients with advanced colorectal cancer. 5-FU acts primarily by inhibiting thymidylate synthase to prevent DNA and RNA synthesis, causing apoptosis in rapidly dividing cancer cells. Emerging literature indicates that 5-FU also suppresses tumor development by activating tumor-reactive T cells. However, the mechanism underlying 5-FU function in T cell activation in human colorectal cancer remains incompletely understood. In a comparative screening of human colorectal tumor tissues, we found that the levels of CD7⁺ cytotoxic T lymphocytes (CTLs) and IL7R⁺ CTLs subsets were significantly lower in human colorectal tumor tissues compared to matched adjacent nonneoplastic colon tissues. As expected, 5-FU therapy effectively suppressed colorectal tumor growth and enhanced total CTLs tumor infiltration in tumor-bearing mice. Analysis of tumor-infiltrating CTLs revealed that 5-FU therapy induced CD7⁺ and IL7R⁺ CTLs differentiation in the mouse colorectal tumor microenvironment. Mechanistically, p53 regulates CD7 and IL7R expression in CTLs by directly binding to the <i>hCD7</i> and <i>hCD127</i> promoters, respectively. Functionally, IL7R⁺CD8⁺ T cells exhibited improved cytotoxicity with elevated expression of T cell effector genes both <i>in vivo</i> and <i>ex vivo</i> in the mouse colorectal tumor microenvironment. More importantly, 5-FU therapy remarkably promoted CD7⁺ and IL7R⁺ CTLs tumor infiltration in colorectal cancer patients, as well as facilitated the infiltration of IL7R⁺ CTLs subsets in colorectal tumor-bearing mice. Overall, our findings determine that colon cancer suppresses the infiltration of CD7⁺ or IL7R⁺ CTLs into tumor microenvironment as a mechanism of immune evasion. 5-FU therapy represses colon cancer progression at least in part through activating p53 to foster CD7⁺ and IL7R⁺ CTLs tumor infiltration, thus proposing a new perspective for the anti-tumor mechanism of 5-FU and a novel, promising therapeutic route by expanding CD7⁺ and IL7R⁺ CTLs to improve the response to 5-FU chemotherapy.</p>