Gene therapy has great potential in treating human diseases, but little progress has been made in preclinical and clinical studies of renal diseases. To find an effective gene delivery approach in the kidney, transparenchymal renal pelvis injection was developed. Using adeno-associated virus serotype 9 (AAV9) vectors, the gene delivery efficiency and safety of this administration method were evaluated. The results showed that the exogenous gene was expressed in the tubular epithelial cells of the injected kidney, with a much lower expression level in the contralateral kidney. Extra-renal transduction in the liver was also observed in this study, with the liver function of AAV9-injected mice comparable to that of control mice. Altogether, the administration of AAV9 vectors by newly established transparenchymal renal pelvis injection achieved the desired exogenous gene expression in renal tubular cells, and hence might be one possible way for gene therapy in renal diseases.