Although 5-fluorouracil (5-FU) is the primary chemotherapy treatment for colorectal cancer (CRC), its efficacy is limited by drug resistance. Ferroptosis activation is a promising treatment for 5-FU-resistant cancer cells; however, potential therapeutic targets remain elusive. This study investigated ferroptosis vulnerability and dihydroorotate dehydrogenase (DHODH) activity using stable, 5-FU-resistant CRC cell lines and xenograft models. Ferroptosis was characterized by measuring malondialdehyde levels, assessing lipid metabolism and peroxidation, and using mitochondrial imaging and assays. DHODH function is investigated through gene knockdown experiments, tumor behavior assays, mitochondrial import reactions, intramitochondrial localization, enzymatic activity analyses, and metabolomics assessments. Intracellular lipid accumulation and mitochondrial DHODH deficiency led to lipid peroxidation overload, weakening the defense system of 5-FU-resistant CRC cells against ferroptosis. DHODH, primarily located within the inner mitochondrial membrane, played a crucial role in driving intracellular pyrimidine biosynthesis and was redistributed to the cytosol in 5-FU-resistant CRC cells. Cytosolic DHODH, like its mitochondrial counterpart, exhibited dihydroorotate catalytic activity and participated in pyrimidine biosynthesis. This amplified intracellular pyrimidine pools, thereby impeding the efficacy of 5-FU treatment through molecular competition. These findings contribute to the understanding of 5-FU resistance mechanisms and suggest that ferroptosis and DHODH are promising therapeutic targets for patients with CRC exhibiting resistance to 5-FU. Intracellular lipid accumulation leads to lipid peroxidation overload, while mitochondrial DHODH deficiency weakens the ferroptosis defense system. This dual vulnerability makes 5-FU-resistant CRC cells susceptible to ferroptosis. Moreover, the redistribution of mitochondrial DHODH to the cytosol increases intracellular pyrimidine pools, impairing the effectiveness of 5-FU through molecular competition. Targeting ferroptosis or DHODH has emerged as a novel therapeutic avenue for treating patients with CRC exhibiting resistance to 5-FU therapies. Display omitted •The susceptibility of 5-FU-resistant CRC to ferroptosis is attributed to the combination of lipid accumulation and mitochondrial DHODH deficiency.•Cytosolic DHODH, like its mitochondrial counterpart, catalyzes dihydroorotate and is involved in pyrimidine biosynthesis.•Ferroptosis and DHODH could be therapeutic targets for 5-FU-resistant CRC patients.