OBJECTIVE： To screen the active fractions of Pseudostellaria heterophylla against myocardial hypoxia/reoxygenation （H/R） injury， and to investigate its mechanism. METHODS： The water extract of P. heterophylla was eluted with D101 macroporous adsorption resin column by water-ethanol gradient elution to obtain water elution fraction （Fr.A）， 30％ ethanol eluting fraction （Fr.B）， 50％ ethanol eluting fraction （Fr.C） and 95％ ethanol eluting fraction （Fr.D）. H/R injury model of rat H9c2 myocardial cells was induced by chemical anoxia method with natrium hydrosulfurosum. MTS assay was used to detect survival rate of cells after pretreated with low-dose， medium-dose and high-dose of each fraction （low-dose， medium-dose and high- dose of Fr.A， Fr.B， Fr.C were 750， 1 500， 3 000 mg/L；low-dose， medium-dose and high-dose of Fr.D were 5， 10， 20 mg/L）， and the strongest active fraction of P. heterophylla was screened. After pretreated with the strongest active fraction， the activity of LDH in cell culture medium， the content of MDA and the activity of SOD in the cells were determined by microplate method， TBA method and WST-1 method， respectively. The apoptotic rate of the cells was detected by flow cytometry. The expressions of Caspase-3， Bcl-2 and Bax were detected by Western blot. RESULTS： After the pretreatment of 4 fractions， the survival rate of cells in Fr.A high-dose group， Fr.B high-dose group and all Fr.C groups were increased significantly， compared with model group （P＜0.05 or P＜0.01）. The activity of Fr.C was the strongest. After pretreated with different doses of Fr.C， the activity of LDH in the culture medium of all groups， the content of MAD， apoptotic rate， the protein expression of Caspase-3 and Bax in medium-dose and high-dose groups were all decreased significantly， compared with model group （P＜0.05 or P＜0.01）； the activity of SOD in high-dose group， the protein expression of Bcl-2 in medium-dose and high-dose groups were increased significantly， compared with model group （P＜0.05）. CONCLUSIONS： The Fr.A， Fr.B and Fr.C of P. heterophylla have anti-H/R injury effects on cardiomyocytes， and the activity of Fr.C is the strongest. Anti-H/R injury of Fr.C may be related to its ability to improve oxidant stress state and scavenge oxygen free radicals， decrease the degree of lipid peroxidation， inhibit cell apoptosis， up-regulate the protein expression of Bcl-2 and down-regulate the protein expression of Caspase-3 and Bax.