Combining different clinical agents to more completely target multiple pathways, including androgen receptor (AR) signalling, in prostate cancer cells is a potentially effective strategy to improve outcomes for men with metastatic disease. We have previously demonstrated that combining sub-effective concentrations of the AR antagonist, bicalutamide, with a histone deacetylase inhibitor, vorinostat, caused marked synergistic death of prostate cancer cells. In this study, we investigated the molecular mechanisms underlying this synergistic action, with the objective of understanding and further enhancing the efficacy of this approach. Expression profiling of LNCaP human prostate cancer cells treated with vorinostat or bicalutamide, alone and in combination, showed that a substantial proportion of the genes modulated by the combination were androgen regulated. Independent pathway analysis identified a number of other pathways and genes, most notably NFKBIA (encoding IkBα, an inhibitor of NF-κB and p53 signalling), as targets of this combinatorial regimen. Depletion of NFKBIA by siRNA knockdown was associated with induction of p53 target genes and enhanced apoptosis of prostate cancer cells, while ectopic overexpression of NFKBIA markedly suppressed cell death induced by the combination of vorinotsat and bicalutamide. These findings implicate this gene as a key mediator of the marked apoptotic action of this combinatorial AR targeting strategy, and a promising new therapeutic target.