In this study, primary murine prostate cancer (PCa) cells were derived using the well-established TRAMP model (transgenic adenocarcinoma mouse prostate). These PCa cells were treated with the histone deacetylase inhibitor (HDACi), valproic acid (VPA), and we demonstrated that VPA treatment has an anti-migrative, anti-invasive and anti-proliferative effect on PCa cells. Using microarray analyses, we discovered several candidate genes that could contribute to the cellular effects we observed. In the present study we could demonstrate that VPA treatment of PCa cells causes the re-expression of cyclin D2, a known regulator that is frequently lost in PCa as we could show using immunohistochemical analyses on PCa specimens. We demonstrate that VPA specifically induces the re-expression of cyclin D2, one of the highly conserved D-type cyclin family members, in several cancer cell lines with weak or no cyclin D2 expression. Interestingly, VPA treatment had no effect in fibroblasts, which typically have high basal levels of cyclin D2 expression. The re-expression of cyclin D2 observed in PCa cells is activated by increased histone acetylation in the promoter region of the ccnd2 gene and represents one underlying molecular mechanism of VPA treatment that inhibits the proliferation of cancer cells. Altogether, our results confirm that VPA is an anticancer therapeutic drug for the treatment of tumours with epigenetically repressed cyclin D2 expression.