Patients with cystic fibrosis (CF) have abnormal concentrations and composition of electrolytes and macromolecules in gastrointestinal secretions. Such alterations could change intestinal surface properties, such as surface hydrophobicity, and may influence the adhesion of macromolecules, bacteria, or microbial toxins to the intestinal surface. The objective of this study was to compare the surface hydrophobicity of the gastrointestinal tract in wild type and CF mice. We used axisymmetric drop shape analysis-contact diameter to determine surface hydrophobicity by measuring contact angles of sessile water droplets placed onto epithelial surfaces. In wild type mice, there were no differences in contact angles between the duodenum, upper jejunum, lower jejunum, and ileum. The contact angle of the gastric mucosa was lower than the rest of the gastrointestinal tract. Contact angles of the proximal colon and distal colon were both higher than that of the gastric mucosa and those of the small intestinal sections. In CF mice, contact angles along the gastrointestinal tract followed the same pattern as in wild type mice. However, contact angles in the ileum and proximal colon of CF mice were greater than those from wild type mice. This study of the murine intestine showed regional differences in surface hydrophobicity comparable to those observed in other mammalian species. In addition, we showed that the ileum and proximal colon of CF mice were more hydrophobic than the corresponding segments in wild type mice. These observations are of potential clinical relevance because patients with CF exhibit clinical manifestations of gastrointestinal disease primarily in the ileum and proximal colon.