The objective of this study was to examine the metabolism of insulin and calcitonin, and their protection by various protease inhibitors, in the large intestine. Fresh caecal contents were prepared from non-fasted rats and the degradation of insulin and calcitonin was studied in a suspension of rat caecal contents, as a model of the content of the large intestine. Both insulin and calcitonin were metabolized in suspensions of rat caecal contents, but the degradation of calcitonin was much faster than that of insulin. The degradation of insulin was fastest at pH 6.8. Protease inhibitors such as camostat and aprotinin inhibited the degradation of insulin and calcitonin in rat caecal contents, which was consistent with the high chymotrypsin activity of these contents. These findings suggest that care should be taken when administering peptide drugs to the large intestine for colon-specific drug delivery because they can be degraded in rat caecal contents. Protease inhibitors might be useful for increasing the stability of these peptides in the large intestine, thereby improving their large-intestinal absorption to the systemic circulation.