Drug resistance is a serious problem in cancer therapy. Growing evidence has shown that docosahexaenoic acid has anti-inflammatory and chemopreventive abilities. Studies have shown that autophagy inhibition and ferroptosis are promising therapeutic strategies for overcoming multidrug resistance. This study was aimed to examine whether docosahexaenoic acid (DHA) could reverse docetaxel resistance in prostate cancer cells. Cell survival was examined by MTT and colony formation. Protein expression was determined by Western blot. Reactive oxygen species (ROS) production was measured by flow cytometry. DHA displayed anti-cancer effects on proliferation, colony formation, migration, apoptosis, autophagy and epithelial mesenchymal transition. Glutathione-S-transferase π is an enzyme that plays an important role in drug resistance. DHA inhibited GSTπ protein expression and induced cytoprotective autophagy by regulating the PI3K/AKT signalling pathway in PC3R cells. DHA combined with PI3K inhibitor (LY294002) enhanced apoptosis by alleviating the expression of LC3B, (pro-) caspase- 3 and (uncleaved) PARP. DHA induced ferroptosis by attenuating the expression of glutathione peroxidase 4 (GPX4) and nuclear erythroid 2-related factor 2 (Nrf2). DHA-treated PC3R cells produced ROS. The ROS and cytotoxicity were reversed by treatment with ferrostatin-1. DHA combined with docetaxel inhibited EMT by regulating the expression of E-cadhein and N-cadherin. In summary, DHA reversed drug resistance and induced cytoprotective autophagy and ferroptosis by regulating the PI3K/AKT/Nrf2/GPX4 signalling pathway in PC3R cells. We propose that DHA could be developed as a chemosensitizer and that the PI3K/AKT /Nrf2/GPX4 signalling pathway might be a promising therapeutic target for overcoming cancer drug resistance.