Objective <p>To elucidate the mechanism through which tumor-associated neutrophil extracellular traps (NETs) contribute to the progression of colorectal cancer (CRC), characterize cellular populations within the CRC tumor microenvironment (TME), and identify potential therapeutic targets.</p> Methods <p>We retrieved the single-cell RNA-seq datasets of CRC from GEO, and performed clustering analysis and subgroup analysis on the quality-controlled single-cell transcriptome data. Subsequently, we interrogated signaling pathways, biological functions, developmental trajectories, survival outcomes, gene regulatory networks, and cellular communication among distinct cell subgroups to delineate tumor heterogeneity during CRC progression.</p> Results <p>Our analyses reveal that the DACH1⁺ and NKD1⁺ CRC subgroups play vital roles in the initiation, progression, and metastasis of CRC. PPIF⁺ neutrophil subgroups promote NETs formation and CRC progression by facilitating mitochondrial reactive oxygen species (mtROS) production. Meanwhile, the C1QC⁺ Mac, RACK1⁺ Tem, RACK1⁺ B, and RACK1⁺ Plasma subgroups exert certain immunosuppressive effects within CRC TME, thus promoting CRC progression. Moreover, RACK1 may serve as a key ecological niche gene in CRC. Furthermore, PPIF⁺ neutrophils modulate the TME via TNFSF14–TNFRSF14 and TNFSF14-LTBR checkpoint axes, thereby sustaining the CRC progression.</p> Conclusion <p>Our research findings have revealed the biological characteristics of CRC under the influence of NETs. Altogether, this study underlines the therapeutic potential value of targeting NETs-related mechanisms in the context of CRC.</p>