Piglets are highly susceptible to gut health-related problems. Intravenously administered chenodeoxycholic acid (CDCA) affects gut health mediated through glucagon-like peptide 2 (GLP-2). To test whether chenodeoxycholic acid is a suitable feed additive for improving gut health, a trial was performed with newly weaned (21 d) piglets offered a diet with or without 60 mg chenodeoxycholic acid/kg feed (n = 24/treatment). Upon weaning, piglets were fasted for 16 h and then intragastrically dosed with 20 g test feed in 40 g water. Subsequently, a jugular blood sample was taken on 45, 90, 135, or 180 min for analysis of GLP-2, peptide YY (PYY), and glucose. Afterwards, piglets were offered the experimental diets ad libitum. On days 3.5, 7.5, and 10.5 after weaning, serum responses to an intragastric dose of lactulose and Co-EDTA were tested at 2 h after dosing in 8 piglets per treatment. Immediately thereafter, piglets were euthanized, intestines were harvested, and permeability was measured ex vivo using the everted gut sac technique with 4 kDa fluorescein isothiocyanato (FITC)-dextran as marker at 25, 50, and 75% of the length of the small intestine. Dietary chenodeoxycholic acid did not affect (P > 0.05) ADFI, ADG, G:F, blood glucose, and plasma GLP-2 and PYY. Serum cobalt and lactulose at day 10.5 tended to be lower in chenodeoxycholic acid pigs compared with control pigs. Serum cobalt and lactulose concentrations were positively correlated (r = 0.67; P <0.01). In conclusion, chenodeoxycholic acid tended to reduce intestinal permeability at 10.5 d after weaning when fed to newly weaned piglets, implying that chenodeoxycholic acid deserves further study as a means for improving intestinal health. The positive correlation found between Co-EDTA and lactulose indicates that both marker molecules measure similar change in permeability.