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Background: Tumor stemness-related genes promote tumor progression and resistance to immunotherapy, but the relation between tumor-infiltrating immune cells and colon adenocarcinoma (COAD) stemness-related genes remains unclear. Methods: The mRNAsi scores, a stemness index derived from transcriptomic data of the TCGA COAD cohort were used for integrated bioinformatics analysis. The Weighted Gene Co-expression Network Analysis (WGCNA) was applied to classify the modules correlated with mRNAsi scores. The Gene Expression Omnibus (GEO) cohorts GSE76402 was used to verify the DEGs between colon cancer and normal tissues. Real-time PCR and Flow-cytometry were used to validate the expression of genes and cancer stem cells marker in the spheres of colon cancer cells. The Single-sample Gene Set Enrichment Analysis (ssGSEA) and CIBERSORT algorithm were used to explore the correlation between significant genes and the immune cells in tumor microenvironment. Result: The mRNAsi score was associated with tumor development and progression. TTK were identified as COAD stemness-related genes based on WGCNA analysis. TTK were negatively correlated with immunity. Interestingly, M1 Macrophages had a higher infiltration in COAD with high TTK expression. Higher infiltration of M1 Macrophages would result in the worse survival rate of COAD patients. Conclusion: TTK may have the potential as a prognostic biomarker and adjuvant target to promote immunotherapy sensitivity and improve outcomes of COAD patients.