BackgroundThe therapeutic targeting of the tumor microenvironment (TME) in colorectal cancer (CRC) has not yet been fully developed and utilized because of the complexity of the cell–cell interactions within the TME. The further exploration of these interactions among tumor-specific clusters would provide more detailed information about these communication networks with potential curative value.MethodsSingle-cell RNA sequencing, spatial transcriptomics, and bulk RNA sequencing datasets were integrated in this study to explore the biological properties of MFAP5 + fibroblasts and their interactions with tumor-infiltrating myeloid cells in colorectal cancer. Immunohistochemistry and multiplex immunohistochemistry were performed to confirm the results of these analyses.ResultsWe profiled heterogeneous single-cell landscapes across 27,414 cells obtained from tumors and adjacent tissues. We mainly focused on the pro-tumorigenic functions of the identified MFAP5 + fibroblasts. We demonstrated that tumor-resident MFAP5 + fibroblasts and myeloid cells (particularly C1QC + macrophages) were positively correlated in both spatial transcriptomics and bulk RNA-seq public cohorts. These cells and their interactions might shape the malignant behavior of CRC. Intercellular interaction analysis suggested that MFAP5 + fibroblasts could reciprocally communicate with C1QC + macrophages and other myeloid cells to remodel unfavorable conditions via MIF/CD74, IL34/CSF1R, and other tumor-promoting signaling pathways.ConclusionOur study has elucidated the underlying pro-tumor mechanisms of tumor-resident MFAP5 + fibroblasts and provided valuable targets for the disruption of their properties.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-023-04281-6.