Globally, the mortality rate of colorectal cancer (CRC) remains high. Despite the development of various treatments, such as targeted therapy and immunotherapy, colorectal cancer continues to be a serious health issue worldwide. Identifying new biomarkers is essential for improving prognosis and tailoring targeted therapies for CRC. This study aims to elucidate the role of CYP4X1 in CRC and its association with patient survival and clinicopathological parameters. Using TCGA databases like GENT2, UALCAN, and GEPIA, we analyzed CYP4X1 expression in CRC and normal tissues. Our analysis revealed a significant increase in CYP4X1 expression in CRC tissues compared to normal tissues. And CYP4X1 high expression was strongly associated with advanced TNM stage, poor tumor differentiation, deeper invasion, and lymph node metastasis. Kaplan–Meier analysis revealed that high CYP4X1 expression correlated with shorter survival times. To investigate the relationship between CYP4X1 expression and colon cancer, WST-1, Transwell, and colony formation assays were performed using colon cancer cells with siRNA-mediated CYP4X1 downregulation. CYP4X1 downregulation significantly inhibited cell proliferation, invasion, migration, and colony formation in vitro. Furthermore, the tumor-forming ability in mice injected with cell lines where CYP4X1 expression was suppressed decreased. In conclusion, CYP4X1 overexpression is closely linked to CRC progression as an independent prognostic marker and potential therapeutic target.