Peptide hormones that modulate insulin secretion have been recognized to have therapeutic potential, with peptides such as amylin (pramlintide acetate, Symlin) and exendin-4 (exenatide, Byetta) now commercially available. Preptin is a peptide that has been shown to increase insulin secretion in vitro and in vivo. Here we describe the first chemical synthesis and biological analysis of a short series of preptin analogues based on the rat preptin sequence, with non-protein amino acid substitutes introduced at Phe 21 to protect this position from proteolytic breakdown. Phe 21 in the preptin sequence was substituted with the non-protein amino acids D-Phe, D-Hphe, 3-aminobenzoic acid, and 1-aminocyclooctane-1-carboxylic acid, which rendered the preptin analogues resistant to chymotryptic protease hydrolysis at this position. Phe 21-substituted preptin was still able to stimulate insulin secretion, with analogues showing a similar dose-dependent effect on insulin secretion from βTC6-F7 mouse β-cells in both the presence and absence of glucose as unmodified rat preptin. Further studies on the stability of the preptin analogues and their effect on insulin secretion are in progress.