Autophagy is a catabolic process for cells that can provide energy sources and allows cancer cells to evade cell death. Therefore, studies on the combination of autophagy inhibitors with drugs are increasing as a new treatment modality in cancer. Previously, we reported the anti-tumor activity of a Palladium (Pd)(II) complex against different types of cancer in vitro and in vivo. Chloroquine (CQ), the worldwide used anti-malarial drug, has recently been focused as a chemosensitizer in cancer treatment. The aim of this study was to investigate the efficacy of a combined treatment of these agents that work through different mechanisms to provide an effective treatment modality for metastatic prostate cancer that is certainly fatal. Metastatic prostate cancer cell lines (PC-3 and LNCaP) were treated with Pd (II) complex, CQ, and their combination. The combination enhanced apoptosis by increasing phosphatidylserine translocation and pro-apoptotic proteins. Apoptosis was confirmed by the use of apoptosis inhibitor. The formation of acidic vesicular organelles (AVOs) was observed by acridine orange staining in fluorescence microscopy. The Pd (II) complex increased AVOs formation in prostate cancer cells and CQ-pretreatment has potentiated this effect. Importantly, treatment with CQ suppressed the pro-survival function of autophagy, which might have contributed to enhanced cytotoxicity. In addition, PI3K/AKT/mTOR-related protein expressions were altered after the combination of treatments. Our results suggest that combination treatment enhances apoptotic cell death possibly via the inhibition of autophagy, and may therefore be regarded as a novel and better approach for the treatment of metastatic prostate cancer. (C) 2020 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.