Objective: CXCR4 is a potential target for cancer gene therapy. In this study, an RNA interference retrovirus vector targeting CXCR4 gene was constructed, and its influence on the invasion of prostate cancer cells by depressing CXCR4 gene expression was analyzed. Methods: the CXCR4-specific siRNA gene was cloned by PCR and inserted into the Pgensil-1 plasmid constaining U6 promoter and EGFP, and then the recombinant fragment was sub-cloned into PLXSN and tested by restriction enzyme and sequencing. The virus obtained from transfected PA317 cells was transfected into prostate cancer cells. The expression of CXCR4 mRNA was detected by RT-PCR. The ability of invasion of prostate cancer cells in vitro was estimated by Transwell experiment. Results: it was showed that with the recombinant PLXSN transfection, the expression of CXCR4 mRNA in prostate cancer cells PC-3m and LNCaP was reduced at rates of (84.26±10.20)% and (88.17±11.23)%, respectively. The results of Transwell experiment also showed that the number of cells under micro-membrane were 14.7±3.1 and 18.9±4.2 in the treated group of PC-3m and LNCaP, respectively, compared with 46.9(5.3 and 66.7±6.0 in the control group (P＜0.05). Conclusion: PLXSN/EGFP-U6-siCXCR4 can significantly depress the expression f CXCR4, by which the invasiveness of prostate cancer cells in vitro was inhibited as well. This recombinant fragment would be helpful in the treatment of prostate cancer.