Objective To discuss the expression of ubiquitinated histone （H2AX） in the prostate cancer （PCa） and adjacent benign prostate tissues， and its relationship with the clinicopathological parameters of the PCa patients，and to clarify the effect of the novel histone deacetylase inhibitor （HDACi） CUDC-101 on the DNA damage， migration， and epithelial-mesenchymal transition （EMT） in PCa. Methods The expression levels of H2AX mRNA in various cancer tissues were retrieved from The Cancer Genome Atlas （TCGA） and UALCAN Databases to analyze the expression differences between PCa and normal prostate tissues and its connection with the clinical prognosis of the patients with PCa； immunohistochemistry method was used to detect the expression of H2AX protein in PCa tissue and adjacent benign prostate tissue， and its relationship with the clinicopathological parameters of the PCa patients was analyzed. The DU145 cells were cultured in vitro and divided into control group， 5% FBS group， 5% FBS+100 μmol·L^-1 CUDC-101 group， and 5% FBS+200 μmol·L^-1 CUDC-101 group.Cell scratch assay and Transwell chamber assay were used to detect the scratch area of the cells and the number of migration cells in the PCa DU145 cells before and after treated with CUDC-101； immunofluorescence staining was used to detect the expressions of epithelial cell marker E-cadherin （E-cadherin） and phosphorylated histone γ-H2AX in the PCa DU145 cells after treated with CUDC-101； Western blotting method was used to detect the expression levels of EMT-related protein， γ-H2AX， and phosphorylated protein kinase B （p-AKT） in the PCa DU145 cells after treated with CUDC-101. Results The TCGA Database and UALCAN Database analysis results showed that H2AX mRNA was highly expressed in the PCa tissue， and the disease-free survival （DFS） of the patients with low expression of H2AX was longer than those patients with high expression of H2AX mRNA （P<0.001）； the immunohistochemistry results showed that compared with adjacent benign prostate tissue，the rate of strong positive expression of H2AX protein in PCa tissue was increased （64.34% vs 14.29%）， and its over-expression was associated with the T stage of PCa （P=0.001） and World Health Organization（WHO）/International Society of Urological Pathology（ISUP） prognostic grapde group（GG） （P=0.004）， but was not associated with the patients’ age， Gleason score， lymphnode metastasis， or nerve and vascular invasion （P>0.05）； the immunofluorescence staining results showed that the compared with control and EMT induction groups，the fluorescence expressions of E-cadherin and γ-H2AX proteins in CUDC-101 treatment group were increased； the cell scratch assay and Transwell chamber assay results showed that compared with control group， the scratch healing area and number of migration DU145 cells in CUDC-101 treatment group was significantly decreased； the Western blotting results showed that compared with EMT induction group， the expression levels of E-cadherin and γ-H2AX proteins in the cells in CUDC-101 treatment group were increased （P<0.05 or P<0.01）， while the expression levels of Vimentin and p-AKT proteins were decreased （P<0.05 or P<0.01）. Conclusion Over-expression of H2AX protein is closely associated with poor prognosis of the patients with PCa. The novel HDACi inhibitor CUDC-101 can regulate the phosphorylations of H2AX and AKT and inhibit the EMT process in the PCa cells.