All-trans retinoic acid (ATRA) has been widely investigated for treatments of many cancers including prostate cancer. HOXB13, silenced in androgen receptor-negative (AR(-)) prostate cancer cells, plays a role in AR(-) prostate cancer cell growth arrest. In this study we intended to elucidate the mechanisms that are involved in the proliferation inhibition of AR(-) prostate cancer cells triggered by ATRA. We discovered that ATRA was able to induce the growth arrest and to increase HOXB13 expression in AR(-) prostate cancer cells. Both EZH2 and DNMT3b participated in the repression of HOXB13 expression through an epigenetic mechanism involving DNA and histone methylation modifications. Specifically, EZH2 recruited DNMT3b to HOXB13 promoter to form a repression complex. Moreover, ATRA could upregulate HOXB13 through decreasing EZH2 and DNMT3b expressions and reducing their interactions with the HOXB13 promoter. Concurrently, the methylation level of the HOXB13 promoter was reduced upon the treatment of ATRA. Results from this study implicated a novel effect of ATRA in inhibition of the growth of AR(-) resistant human prostate cancer cells through alteration of HOXB13 expression as a result of epigenetic modifications.