To study the chemical constituents and the effect of petroleum ether extracts on P-gp in Vitro. Compounds were isolated by methods of phase silica gel column chromatography, gel column chromatography, preparation of thin layer chromatography and various chromatographic techniques, and identified by the physicochemical data of spectral analysis. We measured P-glycoprotein(P-gp) mediated ATP hydrolysis to determine the substrates of P-gp. Ten compounds were isolated from the petroleum ether extracts of semen Euphorbia, and their structures were identified as, 5,15-O-diacetyl-3-O-benzoyl-lathyol(I),5,15-O-diacetyl-3,7-O-dibenzoyl-7-hydroxylathyrol(II), 15-Odiacetyl-3-O-phenyl-6(17)-epoxylathyrol(III), Euphorbia factorL8 (IV),5,15,17 -O-triacety-3-O-benzoyl-17 hydroxyisolathyrol(V), daucosterol(VI),esculetin(VII), Glycerol-1-monooleate(VIII), salicylic acid(IX), oleic acid(X). Much ATP was used by EFL2 and EFL3. For the first time, compounds IX is obtained from the plant. The compounds Ⅰ and Ⅱ were first found may be the substrates of P-gp, and they exhibited moderate activity inhibiting the effluxing function of P-gp.