Angiotensin-I-converting enzyme (ACE) inhibitory peptides play an important role in the prevention and treatment of hypertension to improve human health, because of their characteristics of safety, no side effects, and easy absorption. The objective was to prepare ACE inhibitory peptides from cottonseed protein using an appropriate protease (e.g., alcalase, protamex, flavourzyme, papain, and bromelain) under optimum conditions. The single-factor experiment was used to optimize hydrolysis conditions. The hydrolysate was then purified to identify sequences of bioactive peptides, and in vitro digestion and absorption stabilities of the hydrolysate was evaluated. It was found that the cottonseed protein hydrolyzed by protamex (1500U/g) at pH 8.0 and 55℃ for 5h resulted in the protein recovery rate of 39.8% and the highest ACE inhibitory activity (93.7%). The cotton protein hydrolysate was then separated into five fractions, in which fraction 4 with the highest inhibitory activity (IC₅₀=220.1μg/mL). Three novel ACE inhibitory peptides were identified from fraction 4:VFNNNPQE, LLSQTPRY and VFPGCPET. LLSQTPRY exhibited the highest ACE inhibitory activity (IC₅₀=105.2μmol/L). After in vitro gastrointestinal digestion and absorption by Caco-2 cell monolayer, the cotton protein hydrolysate retained ACE inhibitory activities of 53.8% and 20.5%, respectively. Therefore, the cottonseed protein hydrolysate prepared by protamex could be potentially used as a functional ingredient to develop antihypertensive food products.