Neurotrophin-3 (NT-3) is a type of neurotrophic factor and regulates neural differentiation, survival, and plasticity in both peripheral and central nervous systems. NT-3 expresses in the adult hippocampal dentate gyrus (DG) and has been reported to be upregulated by stress. However, the detailed function of NT3 in the hippocampal DG is unknown. Therefore, we tried to clarify the function of NT3 by overexpressing NT-3 in the hippocampal DG. We generated NT-3 overexpressing mice in the hippocampus by administering adeno-associated virus carrying NT-3 gene. Expression of NT-3 mRNA in the hippocampus was higher more than 35-fold than in the control group. The expression of calbindin, a mature neuronal marker, and the number of FosB positive cells were increased in mature neurons by NT3 overexpression. Moreover, the number of proliferating cells and immature neurons in the hippocampal dentate gyrus was decreased by NT-3 overexpression. In addition, the expression of Vegfd, a factor regulating neurogenesis, was decreased. These results showed that high NT-3 levels in the hippocampus regulate the activation of mature DG neurons and suppress the early phase of neurogenic processes.