To study chemical constituents and their cytotoxic activity from the stems of Calotropis gigantea,ten compounds were isolated from the ethanol extract of C. gigantea by using various chromatographic techniques including silica gel,Sephadex LH-20,etc.Their structures were elucidated on the basis of spectroscopic data including ¹H NMR,¹³C NMR and MS analysis as (-)-pinoresinol-4-O-β-D-glucoside (1),(+)-pinoresinol 4-O-［6′′-O-vanilloyl］-β-D-glucopyranoside (2),(-)-jatrointelignan B (3),(+)-(7R,7′R,7′′S,7′′′S,8S,8′S,8′′S,8′′′S)-4′′,4′′′-dihydroxy-3,3′,3′′,3′′′,5,5′-hexamethoxy-7,9′;7′,9-diepoxy-4,8′′;4′,8′′′-bisoxy-8,8′-dineolignan-7′′,7′′′,9′′,9′′′-tetraol (4),frugoside (5),(+)-dehydrovomifoliol (6),1H-indole-3-carboxylic acid (7),2,3-dimethoxybenzoic acid (8),p-hydroxybenzoic acid (9),and vanillic acid (10).Compounds 1,3,4 and 6-8 were identified from this plant for the first time.All isolated compounds were evaluated in vitro by MTT assay against chronic myeloid leukemia cell K562,human gastric cancer cell SGC-7901,human lung cancer cell A549 and human cervical cancer cell line HeLa.Compounds 4 and 5 exhibited cytotoxic activity against five human cancer cell lines with IC₅₀ values ranging from 12.37 to 49.29 μM and from 0.29 to 0.98 μM,respectively.