This study aimed to identify the bioactive peptides of protamex-pepsin hydrolysate obtained from Paralichthys olivaceus (POppH) and to investigate their potential vasorelaxation and blood pressure regulatory properties, both in vitro and in vivo. The effects of the peptides on endothelium-derived relaxing factors (EDRFs), such as nitric oxide and hydrogen sulfate levels, were analyzed in EA.hy926 cells, and the vasorelaxation-related PI3K/AKT/eNOS pathway activity was analyzed in EA. hy926 cells. An in vitro study revealed that POppH and leucine-glutamic acid-arginine (IER) peptide did not show any cytotoxic effects and significantly increased EDRF production via the regulation of vasorelaxation-associated PI3K/AKT/eNOS protein expression in EA.hy926 cells. An in vivo study suggested that oral administration of IER reduced systolic, diastolic, and mean arterial blood pressure in a spontaneously hypertensive rat model. The bioactive peptide IER derived from Paralichthys olivaceus could be used in the development of functional food products with blood pressure regulatory properties.