PC-3 and DU-145 cells were treated with 20 μM costunolide, 200 nM doxorubicin, or both in the absence or presence ROS scavenger (NAC, 5 mM), JNK inhibitor (SP600125, 10 μM) and P38 inhibitor (SB203580. 10 μM). Using CCK-8 assay to detect the cell viability in PC-3 (A) and DU-145 (B) cells, we found that percentage of survived cells in the CTN + DOX group statistically significant decrease (*p < 0.05, compared with CTN + DOX group), but this observation was reversed significantly when we added the JNK inhibitor (SP600125), P38 inhibitor (SB203580), ROS scavenger (NAC) to the CTN + DOX group (*p < 0.05, compared with CTN + DOX group). Using the caspase-3 activity assay kit to detect the apoptosis in PC-3 (C) and DU-145 (D) cells, we found that caspase-3 activity increased significantly in the CTN + DOX group compared with either agent alone group (*p < 0.05, compared with CTN + DOX group), but this observation was reversed significantly when we added the JNK inhibitor (SP600125), P38 inhibitor (SB203580), ROS scavenger (NAC) to the CTN + DOX. (*p < 0.05, compared with CTN + DOX group). Data presented as mean ± SD were representative of three independent experiments.