This study aims to investigate the chemical constituents from Nelumbinis Plumula and their protective effects on cardiac muscle cell.Fourteen compounds were isolated and purified from 80% ethanol extract by various column chromatography techniques,and identified by ¹H NMR,¹³C NMR and other spectroscopic techniques.The compounds were screened for cardiomyocyte protective activity.Fourteen compounds were identified as naringin (1),kaempferol-3-O-β-D-rutinoside (2),apigenin-6-C-β-L-arabinopyranosyl-8-C-β-D-glucopyranoside (3),luteolin-7-O-β-D-neohesperidoside (4),luteolin-3′-O-β-D-glucopyranoside (5),apigenin-7-O-β-D-glucopyranoside (6),hesperidin (7),isoschaftoside (8),pinoresinol-4-O-β-D-glucopyranoside (9), p-coumaric acid-4-O-β-D-glucopyranoside (10), p-coumaric acid (11),phenethyl-6-O-α-L-arabinfuranosyl-β-D-glucopyranoside (12),icariside D1 (13),icariside F2 (14).Compounds 1-6,9,and 11-14 were firstly isolated from Nelumbinis Plumula.Flavonoids compounds 1-8 showed significant protective effects against hypoxia/reoxygenation injury in HL-1 cardiomyocytes,as well as promoting effects on STAT3 expression in HL-1-STAT3-Luc cells in dose-dependent manner.