Colorectal cancer (CRC) is the third leading cancer globally. Metagenomics has been widely used to analyze the association between the gut microbiota and CRC based on bacterial genus- or species-level comparisons, providing evidence of dysbiosis in CRC development. However, this kind of analysis is unable to provide strain-level information for understanding the individual role of a species in CRC. Here, we used culturomics to isolate CRC mucosal samples and selected 158 Escherichia coli strains to reveal their differences in both genomics and functions by means of phylogenetic analysis and inflammatory induction based on cell and animal experiments. Through genomic comparison, these strains were divided into five phylogroups. The representative strains of each phylogroup significantly induced different levels of cytokine secretion by human leukemic monocyte (THP-1 cell)-based Transwell and animal experiments. Further bioinformatic analysis revealed different profiles of single-nucleotide polymorphisms, genes, and metabolic pathways in the different phylogroups, which can improve the current understanding of the phenotypic differences between these strains. The strain differences revealed in both genomics and functions indicate that the microbiota's function at the strain level should be investigated in order to understand the interacting mechanisms between hosts and gut bacteria.