Tumor hypoxia is a common feature of several cancers, including prostate cancer, and is associated with tumor progression, acquisition of anti-apoptotic potential and therapeutic resistance. We explored hypoxia-inducible genes and examined the effect of knockdown of a target molecule with small interference RNA (siRNA) on the proliferation of human prostate cancer cells. Human prostate cancer cell lines (LNCaP and PC-3) were cultured in normoxia (21% O2) or hypoxia (0.5% O2). Hypoxia-inducible genes were identified by cDNA microarray analysis. Metallothionein (MT) expression was assessed by real-time RT-PCR, Western blot analysis and immunohistochemical staining. siRNA was transfected to knock down MT expression, and the cell cycle and apoptosis were evaluated by flow cytometry analysis. In cDNA microarray analysis, 22 genes (including MT) were up-regulated under hypoxia. MT-1X and MT-2A were up-regulated in real-time RT-PCR. In particular, MT-2A was increased 3-fold in LNCaP and 8-fold in PC-3. The siRNA-MT-2A treatment resulted in a 20% inhibition of cell growth and induced apoptosis in both LNCaP and PC-3. In human prostate tissue, intense staining of MT was observed in cancer cells and residual cancer cells after androgen ablation therapy, while normal tissue was only stained in patches. In conclusion, MT was up-regulated under hypoxia in prostate cancer cells and overexpressed in prostate cancer tissue and residual cancer cells after androgen ablation therapy. As down-regulation of MT by siRNA inhibited cell growth and induced cell death, MT may be a new molecular target for the treatment of human prostate cancer.