<p>Cellular dormancy in colorectal cancer (CRC) significantly contributes to therapeutic resistance, tumor recurrence, and metastasis, resulting in poor prognosis. However, the underlying molecular mechanisms remain poorly understood. Here, we used spheroid culture combined with serum deprivation to enrich and identify dormancy-like CRC cells in vitro and characterized their dormancy-like phenotype by G0/G1 phase arrest, suppressed proliferation and radio-resistance. Compared to proliferative cells, dormancy-like CRC cells maintained similar tumor formation capacity but displayed higher PD-L1 expression level and enhanced migratory ability, indicating greater aggressiveness. Proteomics analysis revealed DDX21 was significantly downregulated in dormancy-like CRC cells, and analysis of clinical data showed an inverse correlation between DDX21 and PD-L1 in CRC patients. This is consistent with the findings that knocking down DDX21 markedly increased PD-L1 levels, suggesting a role for DDX21 in immune evasion. Importantly, overexpressing DDX21 reversed the radio-resistance of dormancy-like CRC cells. Mechanistically, DDX21 downregulation induced a dormancy-like phenotype via p38MAPK activation and AKT suppression to inhibit cellular growth. Furthermore, DDX21 bound to the <i>NUCKS1</i> promoter, and its downregulation reduced NUCKS1 transcription, leading to elevated p27/p21 levels, which reinforced G0/G1 arrest. We also identified HERC2 as the E3 ligase mediating DDX21 degradation via K48/K63-linked polyubiquitination, dependent on the DDX21 helicase core domain. In conclusion, our findings establish DDX21 as a crucial regulator of dormancy-like phenotype in colorectal cancer cells and highlight its potential as a biomarker for dormancy-like tumor populations, radioresistance and poor clinical outcomes in CRC.</p>