At present, there is no effective treatment for prostate cancer (PCa). Previous studies have reported that miR-34a is significantly downregulated in PCa cells; therefore, modulation of miR-34a expression might be a promising therapeutic approach for PCa treatment. To this end, we first verified the downregulation of miR-34a in prostate tumors from a transgenic adenocarcinoma mouse prostate (TRAMP) model. We found that miR-34a overexpression significantly inhibited the cell cycle, viability, and migration of PCa cells by targeting its downstream genes. Next, we tested the concept of intraprostatic injection of rAAV9·pri-miR-34a into 8-week-old TRAMP mice to inhibit PCa progression. We observed that the treatment lowered body weights significantly compared to the control treatment starting at 30 weeks after injection. rAAV9·pri-miR-34a treatment also obviously extended the lifespan of TRAMP mice. Moreover, we confirmed that the neoplasia in the treated prostates was significantly diminished compared to that in the control group. In addition, overexpressed miR-34a downregulated the expression of its target genes. Taken together, our results demonstrated, for the first time, the potential of rAAV-mediated efficient modulation of miR-34a expression in the prostate to inhibit PCa progression by regulating its downstream gene expression.