This study was performed to investigate the angiotensin I-converting enzyme (ACE) inhibitory activity of peptides derived from fertilized eggs and elucidate the inhibition mechanism of these peptides. During incubation, ACE inhibitory activity of the peptides remained stable before day 12 and then increased markedly on day 15. Two ACE inhibitory peptides, VGVIKAVDKKAGGAGKVT and HLFGPPGKKDPV, were purified from peptides on day 15 by consecutive chromatography. Because HLFGPPGKKDPV possessed a higher ACE inhibitory activity (IC50 = 125 μM), an antihypertensive effect of this peptide was further evaluated in vivo. The result showed that this peptide had an antihypertensive effect in spontaneously hypertensive rats (SHRs) at a dosage of 10 mg/kg. Furthermore, Lineweaver-Burk plots suggested that HLFGPPGKKDPV played as a non-competitive inhibitor against ACE, as supported by docking simulation. These data indicated that a fertilized egg has potential as antihypertensive components in functional foods and nutraceuticals.