Purpose <p>5-Fluorouracil (5FU) is a primary chemotherapy for colorectal cancer (CRC), but resistance reduces its effectiveness. HADHB, important in mitochondrial fatty acid β-oxidation, is linked to tumor metabolism changes in various cancers. Its potential influence on 5FU sensitivity in CRC remains unclear. This study aims to elucidate the role of HADHB in modulating 5FU sensitivity in CRC.</p> Methods <p>Collect CRC tissue samples treated with 5FU and perform immunohistochemical staining to evaluate the relationship between HADHB expression and 5FU efficacy. We assessed the impact of HADHB on 5FU IC₅₀ in CRC cells via CCK-8, confirmed HADHB-DUOX2 interaction through co-IP, and used fluorescence staining and flow cytometry to measure ROS levels. Metabolomics and transcriptomics were employed to investigate DUOX2-related metabolic pathways.</p> Results <p>HADHB was significantly upregulated in 5FU-resistant CRC tissues compared to sensitive ones. HADHB knockdown in CRC cell lines improved 5FU sensitivity, increased apoptosis, and caused cell cycle arrest. We identified DUOX2 as a novel HADHB-interacting protein, with their protein levels showing strong positive correlation. Silencing either HADHB or DUOX2 can result in a decrease in ROS production, while DUOX2 overexpression reversed the ROS reduction caused by HADHB knockdown, thereby establishing a functional connection between these two elements in the regulation of ROS. This mechanism may play a crucial role in modulating the sensitivity to 5FU mediated by HADHB.</p> Conclusion <p>HADHB overexpression is linked to 5FU resistance in CRC, indicating it as a potential therapeutic target, likely via the HADHB-DUOX2-ROS pathway.</p>