<p>Resistance to 5-fluorouracil (5-FU) remains a major obstacle to effective chemotherapy for colorectal cancer. Beyond its antimetabolite activity, 5-FU induces oxidative stress that contributes to cytotoxicity; however, molecular determinants underlying differential 5-FU responsiveness remain incompletely defined. Using parental and 5-FU–resistant human colorectal cancer cell models, we identified marked upregulation of calneuron 1 (CALN1), a calcium-binding protein, in resistant cells by RNA sequencing with validation at the mRNA and protein levels. Resistant cells displayed elevated basal cytosolic and mitochondrial Ca²⁺ levels accompanied by increased intracellular and mitochondria-associated reactive oxygen species (ROS) relative to parental cells. In 5-FU–resistant cells, CALN1 depletion exacerbated cytosolic and mitochondrial Ca²⁺ overload and ROS accumulation and increased apoptotic responses under drug-free conditions; 5-FU exposure further enhanced apoptotic responses in CALN1-depleted resistant cells. Pharmacological rescue experiments suggested distinct relationships between Ca²⁺ and ROS responses: the Ca²⁺ chelator BAPTA-AM reduced cytosolic Ca²⁺ and attenuated ROS accumulation and apoptosis under 5-FU exposure, whereas the ROS scavenger N-acetylcysteine reduced oxidative stress without altering Ca²⁺ overload. These findings are consistent with a model in which Ca²⁺ dysregulation contributes to ROS amplification when CALN1 is reduced. Conversely, CALN1 restoration re-established Ca²⁺ homeostasis and improved cell viability under 5-FU exposure. In parental cells, CALN1 overexpression also modestly but significantly attenuated 5-FU-induced loss of viability. Collectively, our results identify CALN1 as a regulator of calcium–redox homeostasis that modulates apoptotic responses to 5-FU in colorectal cancer cells.</p> Graphical abstract <p></p>