The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti-neoplastic agents of natural product origin. We previously reported anti-proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC-3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase-3-dependent pathway that is activated due to dysregulated mitochondrial function. DPT-treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca surge, increased mitochondrial membrane potential (MMP, ΔΨm), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase-3 activation, which in turn induced apoptosis. The antioxidant N-acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca surge, on the other hand the Ca2+ chelator BAPTA inhibited the Ca overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca2+ flux. This suggested that both ROS and Ca signaling play roles in the increased MMP via Ca-dependent and/or -independent mechanisms, since ΔΨm elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS- and Ca-activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca2+ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013. © 2012 Wiley Periodicals, Inc.