The mucus layer in the small intestine is generally regarded as a barrier to drug absorption. However, the mucus layer is a complex system, and presently, only a few studies have been conducted to elucidate its physicochemical properties. The current study hypothesizes that the mucus layer contains solubility-enhancing surfactants and thus might aid the oral absorption of poorly water-soluble drugs. Mucus was sampled from sections of the small intestine of fasted rats to analyze the rheological properties and determine the mucus pH and concentrations of proteins and endogenous surfactants, i.e., bile salts, polar lipids, and neutral lipids. The mucus layer in the two proximal sections of the small intestine exhibited different rheological properties such as higher zero-shear viscosity and lower loss tangent and higher protein concentrations compared to all subsequent sections of the small intestine. The pH of the mucus layer was stable at ~ 6.5 throughout most of the small intestine, but increased to 7.5 in the ileum. The bile salt concentrations increased from the duodenum (16.0 ± 2.2 mM) until the mid jejunum (55.1 ± 9.5 mM), whereas the concentrations of polar lipids and neutral lipids decreased from the duodenum (17.4 ± 2.2 mM and 37.8 ± 1.6 mM, respectively) until the ileum (4.8 ± 0.4 mM and 10.7 ± 1.1 mM, respectively). In conclusion, the mucus layer of the rat small intestine contains endogenous surfactants at levels that might benefit solubilization and absorption of orally administered poorly water-soluble drugs. Graphical Abstract: (Figure presented.)