Objective：To study the treatment effects of androgen receptor（AR）antagonist combined with class I histone deacetylase（HDAC）inhibitors on prostate tumorosphere，and to investigate the possible molecular mechanisms involved in the process. Methods：Prostate cancer LNCaP and 22RV1 cells were cultured in serum-free suspension condition，and the obtained two tumorosphere stem-like cells were treated with AR antagonist MDV3100 with or without the presence of HDAC class I inhibitor MS-275 to study the morphology change and the ability of cell clone formation in monolayer culture condition. Then，quantitative real-time fluorescence polymerase chain reaction（qRT-PCR）was utilized to analyze cancer stem cell marker CD133 expression，and Western blot was used to detect the levels of DNA damage marker H2A.X（p-H2A.X），the cleavage of poly ADP-ribose polymerase（PARP），β-catenin，proto-oncogene c-Myc and cyclin D1. Results：Treatment with MDV3100 alone inhibited CD133 expression in tumorosphere cells. Combination treatment of MDV3100 with MS-275 reduced the number of tumorosphere，inhibited CD133 transcription，enhanced the level of both PARP cleavage and p-H2A.X，decreased expression of β-catenin，c-Myc and cyclin D1. Conclusion：Compared with the treatment of MDV3100 alone，combination treatment of MDV3100 with MS-275 significantly inhibited cancer stem-like tumorosphere cell formation and promoted apoptotic cell death. The drugs-reduced over activation of Wnt/β-catenin/c-Myc/cyclin D1 pathway was possibly involved in the antitumor process. These results provide guidance for clinical application of MDV3100 and MS-275 in prostate cancer management of personal medicine.