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Background Colon cancer remains a significant global health challenge with suboptimal treatment outcomes. While immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, their efficacy in colon cancer is limited. This study investigates a novel combination of cordycepin and CTLA-4 blockade to enhance antitumor immunity through gut microbiota modulation in MC38 colon cancer models.Methods Using a multi-omics approach, we demonstrate that combination therapy significantly inhibited tumor growth ( P<0.01) and improved survival compared to monotherapies. 16S rRNA sequencing revealed specific enrichment of Eubacterium brachy group (3.2-fold increase, P<0.05), which was functionally validated to enhance CD8+ T cell infiltration (2.1-fold, P<0.01) and M1 macrophage polarization (P<0.0001), while reducing immunosuppressive Tregs (43% decrease, P<0.05).Metabolomic profiling identified activation of the histidine metabolism pathway (5.3-fold elevation of Cetirizine N-Oxide, P<0.001), suggesting microbial-metabolic crosstalk in therapeutic efficacy. Single-cell RNA sequencing demonstrated that the triple therapy (cordycepin/CTLA-4/E. rectale) enhanced tumor antigen-specific CD8+ T cell responses, with increased effector molecules (Gzma/b, Ifng) and reduced exhaustion markers (67% decrease in Pdcd1). Notably, enriched CD8+ T cell gene signatures (Rps2, Rps20) correlated with improved progression-free survival in clinical datasets (r=0.56, P<0.001).Results Mechanistically, the combination therapy: (1) promoted conventional CD4 + T cell function while preventing exhaustion, (2) suppressed Treg activity through Bcl6 network inhibition, and (3) disrupted myeloid-Treg immunosuppressive interactions (Cxcl16-Cxcr6, Cd274-Pdcd1; P<0.01). These coordinated effects created a favorable immune microenvironment for tumor control.Our findings establish a novel ‘microbiota-immune’ paradigm where cordycepin potentiates CTLA-4 blockade through: (i) selective enrichment of immunostimulatory gut microbes, (ii) metabolic reprogramming of the tumor microenvironment, and (iii) synergistic modulation of both adaptive and innate immunity.Conclusions Our results suggest that the triple-target strategy of cordycepin, CTLA-4 inhibition, and Eubacterium rectale represents a translatable approach to overcome immune checkpoint inhibitor (ICI) resistance in colorectal cancer with significant potential for clinical development.