Objective To construct a lentiviral vector for RNA interference of the human PFKFB4 gene and establish a PFKFB4 gene silencing on human prostate cancer cells. Methods According to the human PFKFB4 sequence archived in the GenBank database, a shRNA targeting PFKFB4 was designed, and cloned into a linear vector containing the green fluorescent protein (GFP) gene to produce a recombinant lentivirus plasmid. The recombinant plasmids and helper plasmids were transfected into 293T cells, and the titer of the virus was determined. PC-3 cells were infected with the constructed lentivirus, and the silencing effect on PFKFB4 was accessed by real-time PCR (RT-PCR). Results The virus titer was calculated to be 1×108 TU/mL, RT-PCR results showed that the PFKFB4 mRNA expression level of the LV3-PFKFB4shRNA group was significantly lower than the negative control group and the blank group. Conclusion A lentivirus vector for RNA interference of human PFKFB4 was successfully constructed and PC-3 cell lines with PFKFB4 gene knockdown were manufactured, which will be useful for future research of PFKFB4 function in prostate cancer cell models.