Chemotherapy is a major therapeutic strategy for patients with cancer. Owing to the severe inflammatory response of chemotherapy, patients experience extreme discomfort during treatment, and this may interrupt treatment completion. The vitamin D3 has a role in anti-inflammation, but no study has explored whether vitamin D3 has beneficial effects on patients undergoing chemotherapy. In this study, we investigated the effect of calcitriol (Vit-D) on inflammatory responses during 5-fluorouracil (5-FU) treatment. Rats were divided into five groups and treated with 1:1 dilution of 5-FU with equal amount of 0.9% saline, 1:3 dilution of 5-FU with 0.9% saline threefold dilution, 5-FU, Vit-D, or 5-FU + Vit-D. A single dose of 15 mg/kg of 5-FU was intravenously administered for 4 h, and the blood biochemical substances and inflammatory cytokines were assessed after the intervention. The 5-FU group had higher AST, ALT, LDH, and CPK levels than those in the 5-FU + Vit-D group. The 5-FU + Vit-D group had a lower TNF-α value than the 5-FU. The IL-6 levels in the 5-FU + Vit-D group were also significantly lower than those in 5-FU. Calcitriol administration during 5-FU therapy can alleviate the production of inflammatory cytokines and liver damage.