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.