Vitamin D has been proposed to directly reduce the symptoms of chemotherapy-induced gastrointestinal mucositis (GM). In the intestine, active vitamin D, 1,25-dihydroxyvitamin D, is governed, at least in part, by the catabolic enzyme, Cyp24a1. This study assessed whether intestinal-specific Cyp24a1 deletion can prevent intestinal injury and microbiome deterioration that is caused by 5-fluorouracil (5-FU) chemotherapy treatment in mice. The Cyp24a1 gene was selectively deleted in intestinal epithelial cells of C57black/6 mice using the Cre-lox system. Eight-week-old male VilCre-Cyp24a1-/- (Cre+) and Cyp24a1fl/fl control (Cre- mice were euthanized 48 hours after receiving a single intraperitoneal (IP) injection of either 450mg/kg 5-FU or saline control (N=8-10). While 5-FU treatment in Cre- mice, caused intestinal injury by markedly reducing duodenum villous height and crypt area (P<0.01), these effects of 5-FU on villous height and crypt area were not observed in Cre+ mice. Similarly, Ki-67 proliferating cells were maintained in the small and large intestines in the Cre+ mice when treated with 5-FU (P<0.05). Tlr-4 mRNA was markedly induced in Cre+ mice irrespective of 5-FU treatment (P<0.001), while other innate immune genes and inflammatory genes remained unchanged. The diversity of the microbiome, measured by Shannon's index, showed no significant changes, although there were marked alterations in the relative abundance and distribution patterns of microbial species at the family level due to 5-FU treatment and intestinal Cyp24a1 deletion. This study suggests that Cyp24a1 in the intestine could be a viable target for preventing or mitigating mucositis caused by chemotherapy, with further research needed to understand the interactions between vitamin D activity, Tlr-4 expression, and the intestinal microbiome.