Abstract: A method for isolation angiotensin-converting enzyme (ACE) inhibitory peptides from Bacillus subtilis SY fermented soybean meal hydrolyzate through ultrafiltration and nanofiltration followed with liquid chromatography (HPLC) separation was established. The small peptides were separated and purified from the ultrafiltrated fermented soybean meal by chromatography. The peptides were characterized by PPSQ-21 amino acid sequencer and MALDI-TOF-TOF/MS and were synthesized by solid phase peptide synthesis (SPPS). Their ACE inhibitory activities were determined by HPLC, and then their ex vivo cardiac performance of aorta ring of Sprague-Dawley Rats were also determined. The results showed that the filtrate of soybean meal after ultrafiltration was separated into 3 fractions F1 (1000~10000 Da), F2 (500~1000 Da), F3 (<500 Da) and the fraction F3 (<500 Da) had the strongest inhibitory activity. Then two ACE inhibitory small peptides, His-Ala-Gly-Arg (HARG) and Cys-Gly-Ala-Ala-Pro (CGAAP) were isolated and purified from the F3, which showed the inhibition rates of 34.99% and 77.79% on the same concentration (2 mg/mL), respectively, and the CGAAP were higher than fraction F3.The vasorelaxation effect of the peptides were also confirmed by investigate the presence of the peptides with stimulated aorta rings of the Sprague-Dawley rats, but the activity intensity was not directly related to ACE inhibitory activity. Experiments have shown that the ultra-filtrate of fermented soy meal (fermented with Bacillus subtilis from Douchi) hydrolyzate could produce the vascular biologically active components, which provided a reference for further in-depth study of biologically active peptides derived from food.