BackgroundCD4+ T cells play an indispensable role in anti-tumor immunity and shaping tumor development. We sought to explore the characteristics of CD4+ T cell marker genes and construct a CD4+ T cell-related prognostic signature for stage III-IV colorectal cancer (CRC) patients. MethodWe combined scRNA and bulk-RNA sequencing to analyze stage III-IV CRC patients and identified the CD4+ T cell marker genes. Unsupervised cluster analysis was performed to divide patients into two clusters. The LASSO and multivariate Cox regression were performed to establish a prognostic-related signature. RT-qpcr and immunofluorescence staining were performed to examine the expression of ANXA2 in CRC tissue. ResultWe found a higher infiltration abundance of activated memory CD4+ T cells was associated with improved prognosis in stage III-IV CRC patients. Patients were divided into two subgroups with distinct clinical and immunological behaviors based on CD4+ T cell marker genes. And then a prognostic signature consisting of six CD4+ T cell marker genes was established, which stratified patients into high- and low-risk groups. Immune spectrum showed that the low-risk group had higher immune cell infiltration than the high-risk group. Furthermore, the risk score of this signature could predict the susceptibility of stage III-IV CRC patients to immune checkpoint inhibitors and chemotherapy drugs. Finally, we validated that ANXA2 was enriched in Tregs and was associated with infiltration of Tregs in CRC tumor microenvironment. ConclusionThe CD4+ T cell-related prognostic signature established in the study can predict the prognosis and the response to immunotherapy in stage III-IV CRC patients. Our findings provide new insights for tumor immunotherapy of advanced CRC patients.