Angiotensin converting enzyme 2 (ACE2) degrades vasoconstricting angiotensin II (Ang II) into angiotensin (1–7), exerting protective effects in cardiovascular system; thus, ACE2 is considered as a novel target for cardiovascular therapy. The objectives of this study were to identify ACE2 up-regulating peptides from pea protein hydrolysate based on activity-guided purification. Pea protein hydrolysate prepared by a combination of thermoase and pepsin was subjected to various chromatographic fractionation; a peptide with the sequence of AKSLSDRFSY was identified as a novel ACE2 up-regulating peptide. AKSLSDRFSY was resistant to pepsin but not pancreatin in an in vitro digestion system. The resulting new sequences after digestion were characterized. Fragments with the sequences of LSDRFS and SDRFSY showed comparable effects in up-regulating ACE2 expression in vascular smooth muscle cells as the parent peptide. Future study will evaluate in vivo efficacy of the characterized pea protein-derived peptides.