Introduction: Pre-clinical studies have indicated that palifermin may be an effective treatment modality for intestinal mucositis, a debilitating complication of cancer chemotherapy. We determined whether palifermin was protective in rats with experimentally induced intestinal mucositis and the applicability of the sucrose breath test (SBT) to monitor palifermin for its efficacy as an anti-mucositis agent. Results: SBT values and sucrase activity were reduced in all 5-FU-treated groups compared with untreated controls (p < 0.05). At 72 h post 5-FU, sucrase activity was higher in rats treated with palifermin compared with 5-FU controls (p < 0.05). Jejunal and ileal villus heights were lower in all 5-FU groups compared with saline controls. Methods: Dark agouti rats (n = 10) were subcutaneously injected with palifermin or vehicle for 3 d after which they were injected with 5-fluorouracil (5-FU) and sacrificed after 72 h. The in vivo SBT and in vitro sucrase assay were used to evaluate small intestinal function and damage. Intestinal disease severity was determined by histological assessment of villus height and crypt depth. Conclusion: The SBT can monitor the ability of palifermin to modify the functional capacity of the small intestine in rats with intestinal mucositis. Further studies are indicated to investigate the prophylactic potential of palifermin against intestinal mucositis.