Colorectal cancer, a leading cause of global cancer-related morbidity and mortality, poses a significant challenge with its incidence rising. 5-fluorouracil (5-FU), a key chemotherapy agent, faces the challenge of drug resistance. Clock genes, which regulate circadian rhythms, are linked to tumor occurrence, progression, and treatment responses, and are often abnormally expressed in many tumors. Ferroptosis, a non-apoptotic form of cell death, plays a role in tumor drug resistance. Recent research indicates that clock genes may influence tumor cells' sensitivity to ferroptosis by regulating cellular metabolism and oxidative stress responses. Through bioinformatics analysis, we identified the clock gene ARNTL2 as a key factor associated with chemoresistance. ARNTL2 was found to be significantly overexpressed in colon cancer, and was closely correlated with poor prognosis. Experimental validation using in vitro and in vivo models demonstrated that ARNTL2 promotes resistance to 5-FU by upregulating SLC7A11, a critical regulator of ferroptosis. Mechanistically, ARNTL2 directly binds to the SLC7A11 promoter and enhances its transcription, while also influencing SLC7A11 mRNA stability through PHGDH. These findings establish the ARNTL2-SLC7A11 axis as an important mechanism driving ferroptosis resistance and chemoresistance in colon cancer. Furthermore, we explored the therapeutic potential of melatonin (Mlt), a circadian-regulating hormone, and discovered that Mlt can degrade ARNTL2 via the ubiquitination-proteasome pathway, thereby downregulating the ARNTL2-SLC7A11 axis. Our results highlight ARNTL2 as a promising biomarker for predicting chemoresistance and prognosis in colon cancer patients. Additionally, the ability of Mlt to enhance chemotherapy sensitivity by targeting the ARNTL2-SLC7A11 axis offers a novel, low-toxicity strategy for improving treatment outcomes. These findings bridge the fields of chronobiology and oncology, providing new insights for precision medicine approaches in colon cancer.