Abstract: Cytotoxicity screening of extracts of Hypericum petiolulatum Hook.f.et Thoms.ex Dyer were carried out by L1?210 cells.Isolation of chemical constituents were carried out in step with for effective part of H.petiolulatum by column chromatography.The chemical structures were identified by physical and spectral data analysis.Six constituents have been isolated and established as 3α-epi-betulinic acid(1),Patuloside A(2),dimethylpaxanthonin(3),Morusignin D(4),quercetin(5)and quercitrin(6)respectively for the first time.In addition,the crude extract,dichloromethane extract and EtOAc extract possessed cytotoxicity activities with IC50 are 11.0,8.5,10.0(μg/mL)for L1 210 cells respectively.