Prostate cancer (PCa) is one of the most prevalent malignancies in men. However, the molecular mechanism controlling the transformation of androgen-dependent PCa (ADPC) to castration-resistant PCa (CRPC) is largely unknown. Androgen receptor (AR) signaling has been reported to play a key role in this process; thus, searching for the novel AR co-activator is important for identifying the mechanism underlying PCa progression. In this study, we focused on the function of mixed lineage leukemia-5 alpha (MLL5 alpha), an epigenetic regulator that exhibits aberrant expression in PCa. MLL5 alpha was the primary expressed form of MLL5 protein in PCa cells and it significantly suppressed proliferation, invasion, and migration in PCa cell lines. Upon stimulation with dihydrotestoster one (DHT), knockdown of MLL5 alpha significantly suppressed N-myc downstream regulated gene 1 (NDRG1) and Kallikrein-related peptidase 3 (KLK3) expression. MLL5 alpha directly bound with AR on the androgen response elements (AREs) and recruited H3K4me3 to the promoters of NDRG1 and KLK3. Downregulation of NDRG1 partially restored the cell invasion and migration suppressed by MLL5 alpha. As evaluated by the proliferation of PCa cells, overexpression of MLL5 alpha synergistically promoted sensitivity to enzalutamide (ENZ) treatment. In PCa patients, MLL5 alpha expression was lower in the high Gleason score (GS) (GS > 7) group than in the low GS (GS < 7) group. In conclusion, suppression of AR/NDRG1 signaling via androgen deprivation therapy (ADT) may be a potential mechanism of CRPC progression. MLL5 alpha significantly suppressed PCa progression by promoting AR/NDRG1 signaling, indicating that regulating MLL5 alpha expression may be a potential treatment approach for patients with advanced PCa.