Objective To establish a method focusing on regulation of CNN3 gene in the rat hippocampus and help to explore the role of CNN3 gene played in the brain physiology and pathology. Methods One cDNA sequence and three shRNAs targeting CNN3 gene were designed and synthesized. The recombinant lentivirus-mediated expressing and three short hairpin RNA (shRNA) vectors targeting CNN3 gene in the rats were constructed with engineering technology. All recombinant vectors were intravenously injected into rats hippocampi guided by stereotaxic apparatus. Western blot was performed to explore the best shRNA and to study the changes of CNN3 gene in the rat hippocampus after transfection with the silence and over-expressed vectors. Results The lentivirus-mediated vector expressing CNN3-OE and three shRNA vectors targeting CNN3 gene were successfully constructed. Within eight weeks after transfection, the vectors of CNN3-OE and three CNN3-shRNAs changed the expression of CNN3 gene in the rat hippocampus, in particular, all the protein levels of calponin-3 encoded by CNN3 gene were significantly down-regulated along with the time, with the highest inhibitory rate of 73.6% in the CNN3-shRNA2 group. Significant up-regulation of calponin-3 protein level by 93.88%, was found only on the 14th day after transfection. Conclusions Lentivirus-mediated vectors of CNN3-OE and CNN3-shRNAs may regulate in vivo the CNN3 gene level in the local brain region of rats via stereotactic injection.The study lays a foundation for disease prevention and treatment in the future.