Dysregulation of T cells is a major limitation for the clinical success of T-cell based cancer immunotherapies, such as immune checkpoint blockade and chimeric antigen receptor (CAR) T cell therapy. Understanding the underlying mechanisms for regulating T cell functions can facilitate designing therapeutic strategies to improve immunotherapies. Here, we report that TRIM21 impairs CD8+ T cell activation and anti-tumor immunity. Mechanistically, TRIM21 catalyzes the K63-linked ubiquitination on PD-1 at K233, leading to stabilization of PD-1 through antagonizing its K48-linked ubiquitination and degradation. Thus, Trim21 knockout (KO) significantly decreases PD-1 expression and enhances the activation of cytotoxic CD8+ T cells, which sensitizes tumors to anti-CTLA-4 immunotherapy. Notably, Trim21 KO anti-CD19 CAR T cells exhibit improved anti-tumor efficacy. These results reveal the molecular mechanism by which TRIM21-mediated K63-linked ubiquitination on PD-1 restrains the activation of CD8+ T cells, highlighting that targeting the TRIM21-PD-1 axis as a potential therapeutic strategy to potentiate cancer immunotherapy.