Chemotherapy might damage intestinal barrier function (IBF). The aim of the study was to evaluate the effect of dietary fiber on IBF of 5-fluorouracil (5-Fu) stressed rats. Thirty Wistar rats after gastrostomy were assigned randomly to one of three groups (10 of each group), Chow, enteral nutrition (EN), or EN+Fiber (2 g/100 ml solution). They kept their diets respectively for 8 days. 5-Fu (75 mg/kg body weight) was injected intraperitoneally on day 4. Urinary recovery ratios of lactulose and mannitol (L%/M%) were measured on day 3 and day 7. The mesenteric lymph nodes were harvested for bacterial translocation (BT). On day 8, small intestine and colon were taken for wet weight, mucosal thickness of both small intestine and colon and villus height of small intestine. The BT rates of Chow and EN+Fiber groups (20% for both) were lower than that of EN group (70%) (P<0.05). The L%/M% of both EN+Fiber (from 0.0265+/-0.0073 to 0.0274+/-0.0068) and Chow groups (from 0.0268+/-0.0039 to 0.0281+/-0. 0044) were unchanged after stress (P>0.05 for both), whereas that of EN group significantly increased (from 0.0289+/-0.0070 to 0.0331+/-0. 0084) (P<0.01). Chow group gained body weight (4.9+/-4.3 g). EN+Fiber group lost less body weight (-3.1+/-3.4 g) than EN group (-6.6+/-5.2 g) (P<0.05). The intestinal structure of Chow and EN+Fiber groups was superior to that of EN group. In conclusion, dietary fiber could protect the IBF of 5-Fu stressed rats.