Glucose-regulated protein 78 [GRP78, also termed binding immunoglobulin heavy chain protein (Bip)] may be involved in cancer progression and metastasis. However, to date there has been minimal investigation into its potential role in human prostate cancer cells. Recent studies have demonstrated that asymmetric small interfering RNA (asiRNA)-mediated gene silencing is more effective and longer-lasting than conventional symmetric siRNA. Thus, the current study aimed to investigate the effects of GRP78-specific asiRNA on human prostate cancer cells. A series of asiRNAs was synthesized and their efficiency in silencing GRP78 expression in PC-3 human prostate cancer cells was evaluated. The effects of knockdown using asiRNAs were compared to those of knockdown using symmetric siRNAs. The effect of GRP78 silencing on PC-3 cell apoptosis and migration, and the possible mechanisms governing these biological processes were examined. Compared with the symmetric siRNA, transfection with the 15 base pair asiRNA (asiGRP78-3) resulted in greater downregulation of GRP78 expression. GRP78 depletion in PC-3 cells resulted in increased apoptosis and decreased migration of these cells. Experiments investigating the underlying mechanisms of these effects revealed that knockdown of GRP78 attenuated protein kinase B activation and decreased the expression of pro-caspase 9, pro-caspase 3 and vimentin. In conclusion, knockdown of GRP78/Bip expression with asymmetric siRNA led to increased prostate cancer cell apoptosis and reduced cellular migration.