In the course of our screening program for novel chemotherapeutic candidates from plants against adult T-cell leukemia/lymphoma, the extracts of Asclepias curassavica L. showed potent activity against MT-1 and MT-2 cells. Therefore, we attempted to isolate their active components. We identified a new cardenolide, 19-dihydrocalactinic acid methyl ester ( 1 ), along with 16 known cardenolides ( 2 – 17 ). Their structures were determined on the basis of spectroscopic data. Almost all of the isolated cardenolides inhibited the growth of both tumor cell lines. All the doubly linked cardenolides ( 11 – 17 ) except for 14 showed more potent activity than the other cardenolides. A comparison of the activities of 11 , 14 and 16 revealed that the presence of hydroxy or acetoxy functional groups at C-16 led to a decrease in the activity. The 50% effective concentration (EC₅₀) value of calotropin ( 11 ) against MT-2 cells was comparable to the potency of the clinical antineoplastic drug doxorubicin. The cytotoxic effect of 11 toward normal mononuclear cells obtained from the peripheral blood (PB-MNCs) was observed at a concentration 6 to 12 times higher than that used to induce growth inhibition against MT-1 and MT-2 cells. The proportions of annexin V-positive cells after 72 h of treatment with 11 were increased, indicating that it significantly induced apoptosis in MT-1 and MT-2 cells in a concentration-dependent manner. Cell cycle experiments demonstrated that 11 arrested MT-1 and MT-2 cells at the G2/M phase. Therefore, compound 11 may be a promising candidate for the treatment of adult T-cell leukemia/lymphoma.