Jolkinolide B, a bioactive diterpene isolated from the roots of Euphorbia fischeriana Steud, has various biological and pharmacological properties. In this study, the cytotoxicity of highly purified jolkinolide B was tested in human chronic myeloid leukemia (K562) and 2 other cell lines (human esophageal carcinoma Eca-109 and human hepatoma HepG2). The results indicate a significant decrease in the proliferation of all the 3 cell lines when treated with jolkinolide B for 24 h; the IC50 value of cytotoxicity was 12.1 μg/mL (for K562 cells), >50.0 μg/mL (for HepG2 cells), and 23.7 μg/mL (for Eca-109 cells). Further study of K562 cells involving fluorescence and transmission electron microscopy revealed characteristic apoptotic features, such as cell shrinkage, membrane blebbing, loss of microvilli, and nuclear condensation. Agarose electrophoresis of genomic DNA showed a typical fragmentation pattern for apoptotic cells. A kinetic cell-cycle analysis demonstrated that the cell cycle was arrested in the G1 phase. All these results suggest that the anti-proliferation effect of jolkinolide B on K562 cells is achieved by arresting the cell cycle in the G1 phase and subsequently inducing apoptosis.