Colorectal cancer (CRC) patients with mismatch repair (MMR)-deficient (dMMR) but not MMR-proficient (pMMR) tend to benefit from immune checkpoint blockade (ICB) therapy. To uncover the rules governing these varied therapeutic responses, we integrated spatial enhanced resolution omics-sequencing (Stereo-seq), single-cell RNA sequencing, and multiplexed imaging analysis to create high-definition spatial maps of tumors from treatment-naïve and ICB-treated CRC patients. Our results identified the spatial organization and immune status of the tumor-stroma boundary as a distinctive feature of dMMR and pMMR CRCs, which associates with ICB response. The physical interactions and abundance of LAMP3+DCs and CXCL13+T cells may shape the ICB-responsive tumor-stroma boundary, whereas CXCL14+cancer-associated fibroblasts tend to remodel extracellular matrix to form a structural barrier in non-responders. Our work therefore points out the importance of the molecular and cellular spatial structures of tumors in ICB response, raising the possibility of reprogramming tumor-stroma boundary for sensitizing immunotherapies in the majority of CRCs.