Spinosyn A (SPA), derived from a soil microorganism, Saccharopolyspora spinosa, and its derivative, LM2I, has potential inhibitory effects on a variety of cancer cells. However, the effects of SPA and LM2I in inhibiting the growth of human colorectal cancer cells and the molecular mechanisms underlying these effects are not fully understood. Cell viability was tested by using a 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) assay and a colony formation assay. On the basis of the IC50 values of SPA and LM2I in seven colorectal cancer (CRC) cell lines, sensitive (HT29 and SW480) and insensitive (SW620 and RKO) cell lines were screened. The GSE2509 and GSE10843 data sets were used to identify 69 differentially expressed genes (DEGs) between sensitive and insensitive cell lines. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interactions (PPI) were performed to elucidate the molecular mechanisms of the DEGs. The hub gene of the DEGs was detected by Western blot analysis and verified using the CRISPR/Cas9 system. Our data indicate that SPA and its derivative LM2I have significant antiproliferative activity in seven colorectal cancer cell lines and colorectal xenograft tumors. On the basis of bioinformatics analysis, it was demonstrated that epidermal growth factor receptor (EGFR) was the hub gene of the DEGs and was associated with the inhibitory effects of SPA and LM2I in CRC cell lines. The study also revealed that SPA and LM2I inhibited the EGFR pathway in vitro and in vivo.