The functional components of anti-myocardial ischemic activity in walnut were studied. The walnuts were squashed and then extracted by 70% ethanol, and ethyl acetate. The ethyl acetate phase was concentrated and then separated by macroporous resin, MCI, and ODS columns. MTT assay and kit method were applied，along with the model of myocardial ischemia/reperfusion injury to detect the anti-ischemic activity of each isolated component. The results showed that 5,10-dihydroxytryptamine and Shepherdine were separated from walnuts for the first time. Moreover, 5,10-dihydroxytryptamine could significantly maintain the viability of myocardial cell and reduce the enzymes (LDH/CK) released by ischemia/reperfusion injury, having significant protective effects on myocardial ischemia/reperfusion injury.