Sodium caprate (C10) has been widely evaluated as an intestinal permeation enhancer for the oral delivery of macromolecules. However, the effect of C10 on the intestinal absorption of peptides with different physicochemical properties and its permeation-enhancing effect remains to be understood. Here, we evaluated the effects of C10 on intestinal absorption in rats with a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GIP-GLP1) dual agonist peptide (LY) and semaglutide with different enzymatic stabilities and self-association behaviors as well as the oral exposure of the LY peptide in minipigs. Furthermore, we investigated the mechanism of action (MoA) of C10 for improving the intestinal absorption of the LY peptide via live imaging of the rat intestinal epithelium and tissue distribution of the LY peptide in minipigs. The LY peptide showed higher proteolytic stability in pancreatin and was a monomer in solution compared to that in semaglutide. C10 increased permeability in the minipig intestinal organoid monolayer to a greater extent for the LY peptide than for semaglutide. In the rat jejunal closed-loop model, C10 increased the absorption of LY peptide better than that of semaglutide, which might be attributed to higher proteolytic stability and permeability of the LY peptide. Using confocal live imaging, we observed that C10 enabled the rapid oral absorption of a model macromolecule (FD4) in the rat intestine. In the duodenum tissues of minipigs, C10 was found to qualitatively reduce the tight junction protein level and allow peptide uptake to the intestinal cells. C10 decreased the transition temperature of the artificial lipid membrane, indicating an increase in membrane fluidity, which is consistent with the above imaging results. These data indicated that the LY's favorable physicochemical properties combined with the effects of C10 on the intestinal mucosa resulted in an ∼2% relative bioavailability in minipigs.