Zinc ions (Zn2+) are known to influence cell survival and proliferation. However the homeostatic regulation of Zn2+and their role in prostate cancer (PC) progression is poorly understood. Therefore the subcellular distribution and uptake of Zn2+in PC cells were investigated. Inductively coupled plasma mass spectroscopy and fluorescent microscopy with the Zn2+-specific fluorescent probe FluoZin-3 were used to quantify total andfreeZn2+, respectively, in the normal prostate epithelial cell line (PNT1A) and three human PC cell lines (PC3, DU145 and LNCaP). The effects of Zn2+treatment on proliferation and survival were measuredin vitrousing MTT assays andin vivousing mouse xenografts. The ability of Zn2+to protect against oxidative stress via a HIF1α-dependent mechanism was investigated using a HIF1α knock-down PC3 model. Our results demonstrate that the total Zn2+concentration in normal PNT1A and PC cells is similar, but PC3 cells contain significantly higher free Zn2+than PNT1A cells (p< 0.01). PNT1A cells can survive better in the presence of high concentrations of Zn2+than PC3 cells. Exposure to 10 µM Zn2+over 72 hours significantly reduces PC3 cell proliferationin vitrobut notin vivo. Zn2+increases PC3 cell survival up to 2.3-fold under oxidative stress, and this protective effect is not seen in PNT1A cells or in a HIF1α-KD PC3 cell model. A state of Zn2+dyshomeostasis exists in PC. HIF1α is an integral component of a Zn2+-dependent protective mechanism present in PC3 cells. This pathway may be clinically significant through its contribution to castrate-resistant PC survival.