The pathophysiology of hypertension involves the interplay among renin-angiotensin system (RAS), oxidative stress and inflammation. Previous studies focused mainly on ACE inhibitory activity; the goals of this study were to characterize ACE inhibitory peptides from chicken and to investigate their potential antioxidative and anti-inflammatory activities. Spent hen muscle protein hydrolysate digested by thermolysin showed an IC₅₀ value of 39.6 ± 2.3 µg/mL. Following HPLC fractionation and LC-MS/MS analysis, a potent ACE inhibitory peptide IWHHT (IC₅₀ = 9.93 ± 0.65 µM) was identified. IWHHT was not resistant to simulated gastrointestinal digestion and produced a mixture of IW and IWH. IWHHT and IWH significantly reduced the expression of vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-alpha (TNF-α)-induced inflammation. IWHHT, IWH and IW also significantly reduced superoxide level in basal oxidation test. The multi-activities of IWHHT and its associated digestion products may enhance beneficial actions against hypertension.