Prostate cancer is a commonly diagnosed cancer and a leading cause of cancer-related deaths. The bromodomain and extra terminal domain (BET) family proteins have emerged as potential therapeutic targets for the treatment of castration-resistant prostate cancer. A series of 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives were designed and synthesized as selective bromodomain containing protein 4 (BRD4) inhibitors. The compounds potently inhibit BRD4(1) with nanomolar IC50 values and exhibit high selectivity over most non-BET subfamily members. One of the representative compounds 36 (Y08060) effectively suppresses cell growth, colony formation, and expression of androgen receptor (AR), AR regulated genes, and MYC in prostate cancer cell lines. In in vivo studies, 36 demonstrates a good PK profile with high oral bioavailability (61.54%) and is a promising lead compound for further prostate cancer drug development.