The aim of this study was to investigate the apoptosis-inducing effect of gambogic acid (GA) on Jurkat cells and its underlying signaling pathway. Apoptosis induced by GA and some inhibitors was assayed by Annexin V/PI doubling staining. The levels of caspase 3, caspase 8 and caspase 9 activated in living Jurkat cells were measured by flow cytometry. The expressions of caspase 3, caspase 9, p-JNK and P38 were detected by Western blot. The results showed that GA induced apoptosis of Jurkat cells in a dose-dependent manner. The positive cell number of activated caspase 3, caspase 8, caspase 9 and the levels of activated caspase 3, caspase 9, p-JNK, P38 increased after Jurkat cells were treated with GA. ROS, CaMKII, caspase 3, caspase 9, MAPKK, JNK1/2 and P38 inhibitors had some significant effect on GA-induced apoptosis. ROS, CaMKII, MAPKK, JNK1/2 and P38 inhibitors decreased the levels of activated caspase 3, caspase 9 by GA.ROS, CaMKII, MAPKK, JNK1/2 inhibitors decreased the levels of p-JNK by GA. ROS, CaMKII, MAPKK, P38 inhibitors decreased the levels of P38 by GA. It is concluded that GA induced apoptosis of Jurkat cells by activated caspases through activating of ROS-CaMKII-MAPKK-JNK/P38 pathway.