Abstract: To investigate the cellular specificity of short-hairpin RNA ( shRNA ) transcription driven by the prostate-specific membrane antigen enhancer/promoter ( PSMAe/p ) in gene therapy for prostate cancer. Methods: The exogenous enhanced green fluorescent protein ( EGFP ) gene and shRNA interference fragments that target EGFP driven by PSMAe/p were cotransfected into prostate cancer LNCap cells using RNA interference technology. Transferrin-polyethylene glycol-polyethyleneimine ( Tf-PEG-PEI ) was used as the gene transfer vector, and prostate cancer PC-3 cells, bladder cancer T-24 cells, human embryonic kidney HEK293 cells were used as the controls. Quantitative real time- polymerase chain reaction ( PCR ) and western blot analysis were used to detect EGFP gene mRNA and protein expression. Results: In the LNCap cell groups, compared with group A ( the cells transfected with pEGFP-C1 ) and group C ( the cells cotransfected with pEGFP-C1 and the empty pPSMAe/p-UPRT plasmid ), EGFP gene mRNA and protein expression in group B  significantly decreased after the LNCap cells were cotransfected with pEGFP-C1 and the interference plasmid pPSMAe/p-shEGFP-poly( A ) ( P < 0.05 ). However, no significant differences were observed between group A and group C ( P > 0.05 ). In the PC-3, T24, and HEK293 cell lines, no significant differences in EGFP gene mRNA and protein expression were observed among the groups. Conclusion: The short-hairpin RNA transcription driven by PSMAe/p in prostate cancer LNCap cells has cellular specificity, which can be used as a strategy in prostate cancer gene therapy.