Deoxypodophyllotoxin (DPT) is a naturally occurring flavolignan isolated from Anthriscus sylvestris. Recently, it has been reported that DPT inhibits tubulin polymerization and induces G2/M cell cycle arrest followed by apoptosis through multiple cellular processes. Despite these findings, details regarding the cellular and molecular mechanisms underlying the DPT-mediated cell death have been poorly understood. To define a mechanism of DPT-mediated cell death response, we examined whether DPT activates signaling pathways for autophagy and apoptosis. We demonstrated that DPT inhibited cell viability and induced apoptosis in prostate cancer cell lines, as evidenced by a mitochondrial membrane potential and expression of apoptosis-related proteins. Reactive oxygen species (ROS), primarily generated from the mitochondria, play an important role in various cellular responses, such as apoptosis and autophagy. DPT significantly triggered mitochondrial ROS, which were detected by MitoSOX, a selective fluorescent dye of mitochondria-derived ROS. Furthermore, DPT induced autophagy through an up-regulation of autophagic biomarkers, including a conversion of microtubule-associated protein 1 light chain 3 - I (LC3-I) into LC3-II and a formation of acidic vesicular organelles. Moreover, mitochondrial ROS promoted AKT-independent autophagy and ERK signaling. The inhibition of autophagy with 3-methyladenine or LC3 knockdown enhanced DPT-induced apoptosis, suggesting that an autophagy plays a protective role in cell survival against apoptotic prostate cancer cells. Additionally, the results from an in vivo xenograft model confirmed that DPT inhibited tumor growth by regulating the apoptosis- and autophagy-related proteins.