This study aimed to analyze the structural properties of VLSTSFPPK (VL-9) as an ACE-inhibitory peptide. Seven new peptide sequences were synthesized chemically using VL-9 sequence as a template. They were including, VLSTSFPPY(VY-9), VLSTSFPPW (VW-9), VLSTSFPPF (VF-9), VLSTSFCPK(VCK-9), VLSTSFYPK(VYK-9), VLSTSFHPK(VHK-9), and STSFPPK(SK-7). The synthetic peptides were investigated for Angiotensin-Converting Enzyme (ACE)-inhibitory activity, pattern and molecular mechanism of inhibition. Comparing the structure of eight synthetic peptides confirmed the significant role of amino acids in C-terminal and antepenultimate position of ACE-inhibitory peptides. Data revealed more significant role of Lys and Tyr than Phe and Trp in the C-terminal of ACE-inhibitory peptides. Pro and Cys in the antepenultimate position exhibited a positive role in interaction with the ACE enzyme. Removal of Val and Leu from the N-terminal of VL-9, following in silico simulated gastrointestinal digestion, resulted in higher ACE-inhibitory activity and stronger binding to the active site of the enzyme.