Ovarian estrogens (mainly 17β estradiol, E2) have been involved in the regulation of the structure of hippocampus, the center of spatial memory. In recent years, high levels of aromatase (AROM), the estrogen synthase, has been localized in hippocampus; and this hippocampus-derived E2 seems to be functional in synaptic plasticity and spatial memory as ovarian E2 does. However, the contribution of ovarian E2 and hippocampal E2 to spatial memory and neural plasticity remains unclear. In this study, AROM-specific RNA interference AAVs (shAROM) were constructed and injected into the hippocampus of control or ovariectomized (OVX) mice. Four weeks later the spatial learning and memory behavior was examined with Morris water maze, the expression of hippocampal Aβ related proteins, selected synaptic proteins and CA1 synapse density, actin polymerization related proteins and CA1 spine density were also examined. The results showed that while OVX and hippocampal shAROM contributed similarly to most of the parameters examined, shAROM induced more increase in BACE1 (amyloidogenic β-secretase), more decrease in neprilysin (Aβ remover) and Profilin-1 (actin polymerization inducer). More importantly, combined OVX and shAROM treatment displayed most significant impairment of spatial learning and memory as well as decrease in synaptic plasticity compared to OVX or shAROM alone. In conclusion, the above results clearly demonstrated the crucial role of hippocampal E2 in the regulation of the structure and function of hippocampus besides ovarian E2, indicating that hippocampal E2 content should also be taken into consideration during estrogenic replacement.