Valproic acid (VPA) is an established drug in the long-term therapy of epilepsy. Recently, VPA has demonstrated antitumor activity as a histone deacetylase (HDAC) inhibitor. In this study, the anticancer properties of VPA on neural crest-derived human tumor cell lines G361 melanoma, U87MG glioblastoma and SKNMC Askin tumor cells were investigated. The effect of VPA on cell growth, apoptotic activity and invasive ability were evaluated. Firstly, VPA induced cell growth inhibition and apoptotic activity, as demonstrated by sulforhodamine B protein assay, annexin V assay and by Western blot analysis for Bcl2 and Bax expression levels, in all three cell lines. In addition, VPA led to a decrease of HDAC-1 protein level, as assessed by Western blot analysis. Treatment with VPA caused a decrease in the invasive ability of all three cell lines. Since the invasion process involves a complex system of tightly regulated proteases, matrix metalloproteinases (MMPs) and their tissue-specific inhibitors (TIMPs), the effect of VPA on MMP and TIMP expressions was analysed. Exposure to VPA resulted in a decrease of MMP2 and MMP9 activity and expression level, as assesssed by gelatin zymography and Western blot analysis. In addition, exposure to VPA led to enhanced expression of TIMP1, as assessed by Western blot. Taken together, our results, besides providing further evidence that VPA may represent a promising therapeutic strategy in cancer treatment, may help in the design of new protocols geared at the treatment of neural crest-derived tumors.