Valproic acid (VPA), one of the histone deacetylase inhibitors, can suppress prostate cancer (PCa) cells epithelial mesenchymal transition (EMT). Transcriptional intermediary factor 1 gamma (TIF1 gamma) which is a vital protein molecule that possesses ubiquitination enzyme activity, can mediate TGF-beta induced EMT. We aimed to investigate the detailed mechanism between VPA and EMT occurrence in PCa cells to clarify the potential mechanism of TIF1 gamma involved. In our vitro experiments, we first investigated the effect of VPA on the expression TIF1 gamma. After TIF1 gamma was knockdown or overexpressed by related lentivirus, EMT of PCa cells were assessed. When TIF1 gamma knockdown or overexpress stable cell line were established, cells were treated with additional VPA, EMT index were detected and functional experiments were also conducted to confirm whether VPA inhibited EMT of PCa cells via TIF1 gamma. The mono-ubiquitination of Smad4 was analyzed simultaneously. In vivo, mice were facilitated with PC3 cells or TIF1 gamma related knockdown or overexpress virus transfected PC3 cells with or without VPA administration. Results showed that in vitro VPA can increase the expression of TIF1 gamma and also induce the increase expression of E-cadherin, and the decrease of N-cadherin and vimentin. Knocking down of TIF1 gamma can effectively block the effect of VPA on EMT and metastasis while overexpression of TIF1 gamma can strengthen its role. In vivo VPA also showed its anti-growth effect including tumor growth and EMT mediated by TIF1 gamma coincide with in vitro experiments. In conclusion, VPA inhibits the EMT in PCa cells via up-regulating the expression of TIF1 gamma and the mono-ubiquitination Smad4. VPA could serve as a promising agent in PCa treatment, with new strategies based on its diverse effects on posttranscriptional regulation.