Spent hen, a major byproduct of the egg industry, is under-utilized while carries a disposal issue that negatively affects the environment. As a rich source of protein, spent hen proteins can be enzymatically transformed into bioactive peptides with enhanced health-beneficial effects. We previously reported that anti-inflammatory and angiotensin converting enzyme (ACE) inhibitory activities of spent hen hydrolysate. Thus, we hypothesized that spent hen muscle protein hydrolysates can reduce blood pressure in spontaneously hypertensive rats, an animal model similar to human essential hypertension. A number of enzymes have been used to prepare and screen the hydrolysates based on in vitro ACE inhibitory activity, and finally a hydrolysate prepared by thermolysin was selected based on its potency and yield. Oral administration of the hydrolysate at two doses (1,000 and 250 mg/kg body weight per day) over 20 days reduced blood pressure significantly. Results showed that administrating the hydrolysate reduced the level of circulating vasoactive components (e.g. angiotensin II) and inflammatory cytokines (e.g. tumor necrosis factor alpha). In addition, the treatment modulated endothelial dysfunction through improving vasodilation and attenuating vascular inflammation and oxidative stress. This study evidenced the antihypertensive potential of spent hen muscle proteins, which will contribute to developing a value-added solution for spent hen utilization.