The therapeutic options for castration resistance prostate cancer (CRPC) are still limited. Natural bioactive compounds were shown to possess pro-death properties in different tumors. We previously reported that δ-tocotrienol (δ-TT) induces apoptosis, paraptosis and autophagy in CRPC cells. Here, we investigated whether δ-TT might exert its activity by impairing mitochondrial functions. We demonstrated that, in PC3 and DU145 cells, δ-TT impairs mitochondrial respiration and structural dynamics. In both cell lines, δ-TT triggers mitochondrial Ca and ROS overload. In PC3 cells, both Ca and ROS mediate the δ-TT-related anticancer activities (decrease of cell viability, apoptosis, paraptosis, autophagy and mitophagy). As expected, in autophagy-defective DU145 cells, Ca overload was involved in δ-TT-induced pro-death effects but not in autophagy and mitophagy. In this cell line, we also demonstrated that ROS overload is not involved in the anticancer activities of δ-TT, supporting a low susceptibility of these cells to ROS-related oxidative stress. Taken together, these data demonstrate that, in CRPC cells, δ-TT triggers cell death by inducing mitochondrial functional and structural impairments, providing novel mechanistic insights in its antitumor activity.Copyright © 2020. Published by Elsevier Inc.