Anti-hypertensive effect has been known as one of the functionalities of natto. It is deduced that natto shows anti-hypertensive effect by inhibiting angiotensin-converting enzyme (ACE), the key enzyme of renin-angiotensin system. Some research results about anti-hypertensive effect of natto have been reported so far, but there are almost no findings about ACE inhibitory substance derived from natto. We purified and identified the substances from protease-treated hikiwari-natto, whoese ACE inhibitory activity was about 1.4 times higher than that of protease-untreated hikiwari-natto. As a result, it was found that ACE inhibitory substances included novel ACE inhibitory peptides such as Ile-Ile, Ile-Asp, Ile-Phe-Tyr, Leu-Phe-Tyr and Leu-Tyr-Tyr. In addition, the extracts of protease-treated hikiwari-natto showed significant reduction in systolic blood pressure when they were orally administered to stroke prone spontaneously hypertensive rats in a single dose (80mg/kg of rat weight).