Three novel angiotensin-I converting enzyme inhibitory peptides were explored from Sipuncula (Phascolosoma esculenta), a seafood with high protein content. Peptides RYDF, YASGR and GNGSGYVSR were obtained by hydrolysis of the water-soluble protein of Sipuncula with pepsin and trypsin. The peptides were purified through gel filtration and reverse-phase high-performance liquid chromatography, and identified by de novo sequencing method of MALDI-TOF. All three peptides are non-competitive inhibitors of angiotensin-I converting enzyme determined by Lineweaver-Burk plots. Their inhibitory IC50 values were 235, 184 and 2911M, respectively. The inhibitory mechanism was well illustrated through molecular docking. The docking results showed that the differences of inhibitory activities of the three peptides were due to the degree of non-covalent bond-based interactions between the peptides and angiotensin-I converting enzyme, especially the hydrogen bonds. The antihypertensive effect of peptides was confirmed by their lowering blood pressure in spontaneously hypertensive rats with the oral administration as 5 mg/kg body weight. Peptide GNGSGYVSR decreased systolic blood pressure 31 mmHg at 2 h after oral administration, and maintained the level till 4 h. Therefore, peptides from Sipuncula can be considered as promising candidates for ACE inhibition and hypertension treatment.