Nitric oxide-donating non-steroidal anti-inflammatory drugs are safer than traditional NSAIDs and inhibit the growth of prostate cancer cells with greater potency than NSAIDs. In vivo, prostate cancer deposits are found in a hypoxic environment which induces resistance to chemotherapy. The aim of this study was to assess the effects and mechanism of action of a NO-NSAID called NO-sulindac on the PC-3 prostate cancer cell line under hypoxic conditions. NO-sulindac was found to have pro-apoptotic, cytotoxic, and anti-invasive effect on PC-3 cells under normoxia and hypoxia. NO-sulindac was significantly more cytotoxic than sulindac at all oxygen levels. The sulindac/linker and NO-releasing subunits both contributed to the cytotoxic effects of NO-sulindac. Resistance of PC-3 cells to NO-sulindac was induced as the oxygen concentration declined. Hypoxia-induced chemoresistance was reversed by knocking-down hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA using RNAi. Nuclear HIF-1alpha levels were upregulated at 0.2% oxygen but reduced by treatment with NO-sulindac, as was Akt phosphorylation. NO-sulindac treatment of hypoxic PC-3 cells transfected with a reporter construct, downregulated activation of the hypoxia response element (HRE) promoter. Co-transfection of PC-3 cells with the HRE promoter reporter construct and myr-Akt (constitutively active Akt) plasmids reversed the NO-sulindac induced reduction in HRE activation. Real-time polymerase chain reaction analysis of hypoxic, NO-sulindac treated PC-3 cells showed downregulation of lysyl oxidase and carbonic anhydrase IX mRNA expression. Collectively, these novel findings demonstrate that NO-sulindac directly inhibits the hypoxia response of PC-3 prostate cancer cells by inhibiting HIF-1alpha translation via the Akt signalling pathway. The ability of NO-sulindac to inhibit tumour adaption to hypoxia has considerable relevance to the future management of prostate cancer with the same cellular properties as PC-3.