Prostate cancer is one of the most commonly diagnosed cancers and the second leading cause of cancer deaths in Americans. The high mortality rate is mainly attributed to the invasiveness and metastasis of advanced prostate cancer. Targeting the molecules involved in metastasis could be an effective mode of treatment for prostate cancer. In this study, the therapeutic potential of siRNA-mediated targeting of matrix metalloproteinase-9 (MMP-9), urokinase plasminogen activator receptor (uPAR), and cathepsin B (CB) in prostate cancer was carried out using single and bi-cistronic siRNA-expressing constructs. Downregulation of MMP-9, uPAR, and CB inhibited matrigel invasion, in vitro angiogenesis and wound-healing migration ability of PC3 and DU145 prostate cancer cell lines. In addition, the siRNA treatments induced apoptosis in the tumor cells as determined by TUNEL and DNA laddering assays. An attempt to elucidate the apoptotic pathway showed the involvement of FAS-mediated activation of caspases-8 and -7. Further, mice with orthotopic prostate tumors treated with siRNA-expressing vectors showed significant inhibition in tumor growth and migration. In conclusion, we report that the siRNA-mediated knockdown of MMP-9, uPAR, and CB inhibits invasiveness and migration of prostate cancer cells and leads to apoptosis both in vitro and in vivo.