Histone deacetylase inhibitors (HDACIs) have potent anti-cancer activity in a variety of cancer models. Understanding the molecular mechanisms involved in the therapeutic responsiveness of HDACI is needed before its clinical application. This study aimed to determine if a potent HDACI, LBH589 (Panobinostat), had differential therapeutic responsiveness towards LNCaP and PC-3 prostate cancer (PCa) cells. The former showed prometaphase arrest with subsequent apoptosis upon LBH589 treatment, while the latter was less sensitive and had late G2 arrest. The LBH589 treatment down-regulated HDAC6 and sustained ERK activation, and contributed to prometaphase arrest. Mechanistically, LBH589 inhibited HDAC6 activity, caused its dissociation from protein phosphatase PP1α, and increased 14-3-3ζ acetylation. Acetylated 14-3-3ζ released its mask effect on serine 259 of c-Raf and serine 216 of Cdc25C subsequent to de-phosphorylation by PP1α, which contributed to ERK activation. Enhanced ERK activity by LBH589 further down-regulated HDAC6 protein levels and sustained ERK activation by free-forward regulation. The sustained Cdc25C and ERK activation resulted in early M-phase (prometaphase) arrest and subsequent apoptosis in the most sensitive LNCaP cells but not in PC-3 cells. This study provides pre-clinical evidence that HDAC6 may serve as a sensitive therapeutic target in the treatment of prostate cancer with HDACI LBH589 for clinical translation. This study also posits a novel mechanism of HDAC6 participation in regulating the c-Raf-PP1-ERK signaling pathway and contributing to M phase cell-cycle transition.