Purpose: Treatment of locally advanced rectal cancer is multimodal and includes total neoadjuvant therapy (TNT) with chemoradiation and chemotherapy. Response to TNT is highly variable and correlates with oncologic outcomes. There are limited biomarkers for rectal cancer radiation response, and there is also a critical need to identify potential therapeutic targets that could improve radiation sensitivity. The gene FDXR encodes a mitochondrial flavoprotein involved in electron transport, iron metabolism and ferroptosis. This study evaluates the role of FDXR in rectal cancer radiation response. Methods: Pretreatment biopsies from 33 rectal cancer patients were analyzed previously, identifying FDXR as differentially expressed among different AJCC Tumor Regression Score (TRS). FDXR expression was examined in public datasets. FDXR expression was measured by RT-qPCR in ten colorectal cancer (CRC) cell lines and correlated with radiosensitivity parameters (IC50, D10, SF2). Stable FDXR knockdown lines (HCT116, SW480) were generated using lentiviral shRNA. Knockdown was confirmed by Western blot. To assess mitochondrial morphology and structural integrity, Transmission electron microscopy (TEM). Clonogenic survival after irradiation, viability (CCK-8), lipid peroxidation (Image-iT), mitochondrial iron (MitoFerroGreen), and ROS levels were assessed before and after irradiation. Results: FDXR was significantly overexpressed in rectal tumors compared with normal tissue in TCGA and GSE87211. In our patient cohort, higher FDXR expression correlated with poorer response (TRS 2-3) and showed strong predictive ability (AUC=0.8577). Across CRC cell lines, FDXR expression correlated positively with IC50, D10, and SF2. FDXR knockdown caused loss of mitochondrial structural integrity on TEM and significantly reduced clonogenic survival while increasing radiation-induced cell death. Knockdown lines exhibited higher mitochondrial iron, increased ROS, and elevated lipid peroxidation. Conclusions: FDXR is a strong biomarker of rectal cancer radiation response. Loss of FDXR enhances ferroptosis through iron accumulation, ROS generation, and lipid peroxidation, increasing radiosensitivity. These results suggest that targeting FDXR could enhance radiation efficacy, offering a novel strategy for radiosensitization in rectal cancer.