Effect of an N-succinyl-L-trialanine p-nitroanilide-hydrolyzing protease (STA-protease) purified from Pronase on glucose metabolism was investigated by an intravenous injection into fasted mice. The maximum decrease in blood sugar level by STA-protease was observed 2 h after the injection of a dose of 5.0 mg/kg. The hypoglycemic activity was observed with other microbial proteases, such as Pronase E and subtilisin BPN', whereas no activity was found with the modified enzymes which almost wholly lost their proteolytic activities by the treatment with diisopropyl fluorophosphate or guanidine HCl. The increase in blood sugar level by epinephrine and the conversion of [1-¹⁴C] pyruvate into blood glucose were distinctly suppressed with STA-protease as well as 5-methoxyindole-2-carboxylic acid (MICA), an inhibitor of gluconeogenesis. A slight increase in glycogen content in diaphragm of mice was observed during 2-4 h after the injection of STA-protease, though it was markedly increased by insulin. In test of glucose tolerance, the increase in blood sugar was distinctly suppressed by insulin but not by STA-protease. Blood lactate level was not subjected to change by STA-protease, in contrast to MICA. Serum insulin level was not affected by STA-protease. Therefore, STA-protease may affect the in vivo metabolism of glucose in a different way from the in vitro action which has been reported to react with cell surface and consequently to mimic the actions of insulin.