The relationship of TMPRSS2-ERG fusion gene with matrix metalloproteinase-9 (MMP-9) and PLXNB1 (plexin B1) in regulation of prostate cancer (PCa) aggressiveness was investigated. Fluorescence in situ hybridization (FISH) assays, qRT-PCR and western blot analysis were employed to detect the expression of TMPRSS2-ERG fusion gene, ERG, MMP-9 and PLXNB1 of 135 human tissues, which included 55 metastatic PCa cases, 50 localized PCa cases and 30 BPH cases. Then using siRNA (anti-ERG, MMP-9 and PLXNB1, respectively) downregulation of the target gene of VCaP and PC-3 cells, MTT and Transwell were performed. The results showed that the positive rate of TMPRSS2-ERG fusion was 38.1% (40/105) in total PCa samples, 47.3% (26/55) of metastatic PCa, 28.0% (14/50) of localized PCa, while 0.0% (0/30) in BPH samples. The mRNA and protein expression of ERG, MMP-9 and PLXNB1 were higher in metastatic PCa (P<0.0001), and the mRNA expression of the three genes were positively correlated with TMPRSS2-ERG fusionin PCa group (P<0.0001). siRNA transfected PCa cells can effectively downregulate the target gene expression, and we identified that MMP-9 and PLXNB1 expression were all regulated by TMPRSS2-ERG fusion gene. While only PLXNB1 contributed to TMPRSS2-ERG mediated enhancements of VCaP cell migration and invasion. The results demonstrated that PLXNB1, but not MMP-9, was the target gene directly related to TMPRSS2-ERG in PCa cell migration and invasion.