Objective To explore the effect of open chromatin state on functional pathways related to colon cancer using the data of assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) from The Cancer Genome Atlas (TCGA) database. Methods ATAC-seq and RNA-seq data of colon cancer were downloaded from TCGA, and the quality of ATAC-seq data was controlled using R 3.5.3 software. The ATAC-seq peaks of all samples were annotated, and the Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out. The key genes of colon cancer, including APC regulator of Wnt signaling pathway (APC), Kirsten rat sarcoma viral oncogene (KRAS) and v-raf murine sarcoma viral oncogene homolog B1 (BRAF), were selected to analyze the correlation between ATAC-seq peaks of the 3 genes and the fragments per kilobase of exon model per million mapped fragments (FPKMs) of RNA-seq. The tissue samples of TNM stage Ⅲ+Ⅳ and Ⅰ+Ⅱ were selected to analyze the differential ATAC-seq peaks, and KEGG pathway enrichment analysis was performed for up-regulated and down-regulated peaks annotated genes. Results The ATAC-seq peaks of colon cancer were evenly distributed in the chromosome, and most of them were located in promoter region (30.17%, 5.42% and 3.88% in the distance from transcription initiation site ≤ 1 kb, >1-2 kb and >2-3 kb, respectively) and distal intergenic region (26.17%), which were consistent with 2 main types of chromatin open region. GO function and KEGG pathway enrichment analyses showed that the peaks annotated genes in ATAC-seq data of colon cancer were significantly enriched in cancer-related functions and pathways, such as intercellular signaling conduction of Wnt signaling pathway and epidermal growth factor receptor (ErbB) signaling pathway. The RNA-seq FPKMs of APC, KRAS and BRAF were positively correlated with ATAC-seq peaks in promoter region. The up-regulated peaks annotated genes were significantly enriched in ErbB signal pathway, Wnt signal pathway, PI3K-Akt signal pathway, p53 signal pathway and other signal pathways related to proliferation, invasion and metastasis, while down-regulated peaks annotated genes were significantly enriched in T cell receptor signaling pathway, B cell receptor signaling pathway, cell adhesion molecule signaling pathway and other immune recognition related signaling pathways in patients at TNM stage Ⅲ+Ⅳthan at stage Ⅰ+Ⅱ. Conclusion Open chromatin plays an important role in the functional pathways related to colon cancer.