Changes in the intracellular level of reactive oxygen species (ROS) including superoxide anion, hydroxyl radical, hydrogen peroxide and finally cellular acid-base equilibrium are reported to play an important role in the early step of apoptosis. All of which would precede the loss of mitochondrial membrane potential and releasing of those apoptotic inducing factors such as cytochrome c as well as caspases activation. Any potential chemotherapeutic agent that could drive such changes in ROS would be particularly attractive. Recently we have reported the potential use of Gleditsia sinensis extract (GSE) in cancer therapy including solid tumour and leukaemia cell lines as well as primary cultured leukaemia cells in vitro. We demonstrated that apoptotic activity is involved. Here we further showed that the mechanism of GSE induced apoptosis, including an early decreasing of intracellular superoxide anion as measured by nitroblue tetrazolium (NBT) reduction assay. This phenomenon readily occurred before any shrinkage of cancer cells including MDA-MB231 breast cancer, CNE-2 nasopharyngeal carcinoma, K-562 chronic myelogenous leukaemia and KG1-a, acute myelogenous leukaemia. Cell viability was determined by morphological investigation and the [3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay. Furthermore, the superoxide dismutase activity from those cellular extracts after GSE treatment seemed to be increased. Taken together, we speculate that the GSE-induced apoptosis, via ROS pathway, involves an early decrease of intracellular superoxide anion.