The objective of the study was to determine the region of maximum permeation of salmon calcitonin (sCT) through the gastrointestinal tract and to investigate the mechanism of permeation. For regional permeability determination, male Sprague-Dawley rats (250–300 g) were anesthetized and the gastrointestinal tissues were isolated. Stomach, duodenum, jejunum, ileum, or colon tissues were mounted on Navicyte side-by-side diffusion apparatus. Salmon calcitonin solutions (50 μM in phosphate-buffered saline, pH 7.4, 37°C) were added to the donor side, and the samples were removed from the receiver compartment and analyzed by competitive radioimmunoassay (RIA). For mechanistic studies, Caco-2 cells were grown on Transwell inserts (0.4-μm pore size, 0.33 cm2 area) in a humidified 37°C incubator (with 5% CO2). Transport experiments were conducted for sCT solutions (50 μM in Dulbecco's modified eagle's medium [DMEM], pH 7.4) from the apical-to-basolateral (A-to-B) direction and B-to-A direction at 37°C and from the A-to-B direction at 4°C. Cell monolayer integrity was monitored by mannitol permeability and transepithelial electrical resistance (TEER) measurements. The permeability coefficients (× 10−9, cm/sec) for sCT through rat stomach, duodenum, jejunum, ileum, and colon tissues were 0.482±0.086, 0.718±0.025, 0.830±0.053, 1.537±0.32, and 0.934±0.15, respectively. The region of maximum sCT permeability is ileum followed by colon, jejunum, duodenum, and stomach. The permeability coefficients (× 10−6, cm/sec) for sCT through Caco-2 cell monolayer were 8.57±2.34 (A-to-B, 37°C), 8.01±1.22 (A-to-B, 4°C), and 6.15±1.97 (B-to-A, 37°C). The mechanism of its permeation is passive diffusion through the mucosa as determined from the Caco-2 monolayer permeability of sCT.