Cystatin E/M (CST6) is a natural inhibitor of lysosomal cysteine proteases. Recent studies have shown that experimental manipulation of CST6 expression alters the metastatic behavior of human breast cancer cells. However, the association of CST6 with prostate cancer invasion and progression remains unclear. Here, we show that CST6 is robustly expressed in normal human prostate epithelium, whereas its expression is downregulated in metastatic prostate cell lines and prostate tumor tissues. Treatment of metastatic prostate cell lines with the histone deacetylase inhibitor trichostatin A resulted in significant induction of CST6 mRNA levels and increased CST6 protein expression, indicating that epigenetic silencing may play a role in the loss of CST6 expression observed in prostate cancer. CST6 overexpression in human prostate cancer cells significantly reduced in vitro cell proliferation and matrigel invasion. Furthermore, the results from a bioluminescence tumor/metastasis model showed that the overexpression of CST6 significantly inhibits tumor growth and the incidence of lung metastasis. These results suggest that the downregulation of the CST6 gene is associated with promoter histone modifications and that this association plays an important role in prostate cancer progression during the invasive and metastatic stages of the disease.