Intestinal injury is one of the main side-effects of cisplatin (CP) chemotherapy, severely limiting the clinical application of CP. Vitamin D₃ is an essential nutrient for mammals and exists in a wide range of foods; it regulates immune function and reduces oxidative stress. However, the effect of vitamin D₃ on CP-induced intestinal injury is not elucidated. This is the first study to investigate the relationship between ferroptosis and the protective effect of vitamin D₃ on CP-induced intestinal injury. An animal model of CP-induced intestinal injury was established to evaluate the effect of vitamin D₃ on CP-induced intestinal injury and elucidate the underlying mechanisms. We found that vitamin D₃ alleviated intestinal barrier injury and the abnormal morphological structure in CP-induced intestinal injury mice. Vitamin D₃ suppressed oxidative stress by increasing the antioxidant capacity, inhibiting the accumulation of ROS and MDA, and reducing intestinal inflammatory responses. Vitamin D₃ also decreased excessive mitochondrial fission and increased mitochondrial ATPase activity by inhibiting ROS production, which further alleviated the accumulation of ROS. We also confirmed the involvement of ferroptosis in CP-induced intestinal injury in our animal model using ferrostatin-1 (Fer-1) intervention. Vitamin D₃ decreased iron accumulation and reversed GPX4 and DHODH down-regulation. In conclusion, vitamin D₃ protected against CP-induced intestinal injury by inhibiting ferroptosis and alleviating oxidative stress and ROS-mediated excessive mitochondrial fission, suggesting that it may be a novel and promising candidate to prevent CP-induced intestinal injury.