Tumor immune evasion is intricately linked to malignant tumor progression and contributes to the failure of anti-cancer immunotherapy. Serine/threonine protein kinase 25 (STK25) has been previously implicated in the progression of various neoplastic diseases. However, the function of STK25 in the colorectal cancer (CRC) microenvironment remains unclear. Here, it is demonstrated that STK25 global knockout (STK25-/-) mice and STK25-knockout tumor-bearing mice exhibited enhanced effectiveness of anti-PD-1 immunotherapy, which leads to significant tumor suppression with increased recruitment of CD8+ T cells. Mechanistically, STK25 deficiency increased PD-L1 protein levels by regulating PD-L1 K48-linked ubiquitination in a NEDD4-dependent manner. Moreover, CRC patients with low STK25 expression are more responsive to immune checkpoint blockade (ICB) therapy compared to those with high STK25 levels. Taken together, the findings reveal a critical role of STK25 for regulating PD-L1 protein stability in tumor immune evasion, and suggest that targeting STK25 may provide a potential approach to increase sensitivity to the ICB treatment in patients with CRC.