Colorectal cancer (CRC) is one of the most lethal and prevalent malignancies. While the overexpression of pioneer factor GATA6 in CRC has been linked with metastasis, its role in genome-wide gene expression dysregulation remains unclear. Through studies of primary human CRC tissues and analysis of the TCGA data, we found that GATA6 preferentially binds at CRC-specific active enhancers, with enrichment at enhancer-promoter loop anchors. GATA6 protein also physically interacts with CTCF, suggesting its critical role in 3D genome organization. The ablation of GATA6 through AID and CRISPR systems severely impaired cancer cell clonogenicity and proliferation. Mechanistically, GATA6 knockout induced global loss of CRC-specific open chromatins and extensive alterations of critical enhancer-promoter interactions for CRC oncogenes. Last, we showed that GATA6 knockout greatly reduced tumor growth and improved survival in mice. Together, we revealed a previously unidentified mechanism by which GATA6 contributes to the pathogenesis of colorectal cancer.