Abstract Pacific oyster (Crassostrea gigas) was hydrolysed by neutral proteinase followed by alkaline proteinase to produce angiotensin-I converting enzyme (ACE) inhibitory peptides. Orthogonal experiment and response surface analysis based on SDS-PAGE and ACE inhibitory activity were used to evaluate the optimal hydrolysis condition. The results showed that the first hydrolysis should be performed at solid to liquid ratio of 1:4 (w:v), pH 7.0, neutral proteinase dosage of 1.2%, with hydrolyzing duration of 60 min. The second hydrolysis was at 53 °C, pH 8.3, alcalase dosage of 0.42%, with hydrolyzing duration of 73 min. High performance liquid chromatography (HPLC) analysis showed that almost all (99.72%) the peptides produced were with molecular weight lower than 3000 Da. Meanwhile, oyster peptides revealed high ACE inhibitory activity, with IC50 of 0.8 mg/mL. The amino acid composition analysis indicated that the content of glutamic acid was the highest and cysteine was the lowest in the peptide products. Among the total amino acids, essential amino acids occupied 36.6% while hydrophobic amino acids accounted for 37.2%. Our present work established a theoretical reference for the development of oyster antihypertensive peptides.