Objective To investigate the antitumor effect of PRDM5 gene in prostate cancer cells. Methods PRDM5 gene was cloned and inserted into lentiviral vector using polymerase chain reaction (PCR), restriction endonuclease and T4 DNA ligase connected method. The lentiviral plasmids carrying PRDM5 gene (LV-PRDM5) or control lentivirus (LV-Luc) were co-transfected with lentiviral packaging plasmid mix into 293T cells by liposome method. The viral supernatants were collected and transduced into human prostate cancer cells 22Rv1. The expression of PRDM5 was verified by Western blotting analysis. The cell proliferation and clone formation ability were detected by cell multiplication and cell cloning experiments. The anchorage independent growth rate of prostate cancer cells was assessed by soft agar colony formation assay. Results The lentivirus vector expressing PRDM5 gene was constructed successfully, and the viral supernatants were obtained. The prostate cancer cell line 22Rv1 stably expressing exogenous PRDM5 was screened and verified by Western blotting analysis. Compared with control cells, the prostate cancer cell line 22Rv1 expressing PRDM5 showed a lower growth rate (multiplication time: [52.5±1.4] vs [44.0±1.3] h), clone formation rate ([1 114±98] vs [1 361±123] colonies per dish) and anchorage independent growth rate ([94.6±8.7] vs [154.0±3.5] colonies per cell, P<0.05). Conclusion Overexpression of PRDM5 has inhibitory effect against proliferation, clone formation and anchorage independent growth of prostate cancer cells in vitro.