Trichosanthin (TCS), a ribosome inactivating protein extracted from the root tuber of a traditional Chinese medicinal herb Trichosanthes kirilowii, possesses anti-tumor and anti-human immunodeficiency virus (HIV) activities. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, 2′, 7′-dichlorofluorescin diacetate (DCFH-DA), Indo-1 and Fluo 3-AM, TCS-induced changes in nuclear morphology, reactive oxygen species (ROS) and intracellular calcium concentration (［Ca²⁺］_i) during the apoptosis of choriocarcinoma cells (JAR) were simultaneously observed for the first time. The results indicated that TCS-induced increase in ［Ca²⁺］_i and ROS formation were involved in apoptosis of JAR cells, and that TCS-induced ROS formation was related to TCS-evoked increase in ［Ca²⁺］_i. Further studies with confocal laser scanning microscopy revealed that TCS-evoked increase ［Ca²⁺］_i was not the main factor responsible for TCS-induced ROS formation, and that TCS might induce the production of ROS through its interaction with membrane-bound receptor.