Peperomia pellucida (L.) Kunth. (Piperaceae) is empirically used in traditional medicine to control blood pressure. The objective of this study was to isolate ACE inhibitor from aerial part of the plant. Using chromatography technique was isolated flavonoid as ACE inhibitor. The chemical structure was determined as 3’,4’, Dihydroxy-3-5-dimethoxy flavone-7-O-β-rhamnose based on the spectral data (UV, IR, LC-MS,1H-NMR,13C-NMR, and 2DNMR) and compared with that reference. In vitro assay of ACE inhibitory activity that showed IC50 of the compound was 7.72 μg/mL. The result of kinetic suggested that the compound inhibit the enzyme activity by competing with the substrate for the active site. The results supported the traditional use of the plant antihypertensive. © 2016, Sphinx Knowledge House. All rights reserved.