5-Fluorouracil (5-FU), in association with leucovorin (LV), is the most used chemotherapy agent in the treatment of colorectal cancer. Response rate, as well as side-effect incidence, increases with the dose intensity of regimens that are used. The most common dose-limiting toxicity for 5-FU/LV modulation is diarrhea. To assess the modification of small intestinal function, we investigated the changes in intestinal permeability (IP) and intestinal absorption (IA) in 41 chemo-naive patients (21 men and 22 women; mean age, 61 ± 9 years) with advanced colorectal cancer after treatment with the association of folinic acid and 5-FU. After chemotherapy administration, we found a marked increase in IP and a reduction in IA, measured as cellobiose–mannitol (CE–MA) ratio (p < 0.0001) and D-xylose absorption (p = 0.0001), respectively. Patients who experienced diarrhea have an increase in CE–MA ratio and a reduction in D-xylose absorption values, both statistically significant. Cellobiose–mannitol ratio and D-xylose absorption tests can be used for the assessment of toxic effect of 5-FU on mature intestinal epithelium and also for evaluating the role of cytoprotective agents.