The potential use of trypsin inhibitors (TI) present in the viscera of Japanese common squid (Todarodes pacificus) was examined for diabetes treatment. In a single-glucose tolerance test, the crude TI (C-TI) fraction significantly stimulated insulin secretion by normal Wistar rats and by diabetic GK rats. The highly purified TI (H-TI) fraction displayed the same effect on the glucose tolerance test with the GK rats. The TI activity of C-TI was not decreased by pepsin or chymotrypsin digestion in vitro, and the C-TI fraction did not have α-glucosidase inhibitory activity. These results indicate that the substance with the stimulating effect on insulin secretion in rats is the squid TI and that the hypoglycemic effect on glucose tolerance in GK rats is related to an improvement of insulin secretion due to the squid TI.