Recombinant adeno-associated viral (rAAV) vector-induced neuropeptide Y (NPY) overexpression in the hippocampus exerts powerful antiepileptic and antiepileptogenic effects in rats. Such gene therapy approach could be a valuable alternative for developing new antiepileptic treatment strategies. Future clinical progress, however, requires more detailed evaluation of possible side effects of this treatment. Until now it has been unknown whether rAAV vector-based NPY overexpression in the hippocampus alters normal synaptic transmission and plasticity, which could disturb learning and memory processing. Here we show, by electrophysiological recordings in CA1 of the hippocampal formation of rats, that hippocampal NPY gene transfer into the intact brain does not affect basal synaptic transmission, but slightly alters short-term synaptic plasticity, most likely via NPY Y2 receptor-mediated mechanisms. In addition, transgene NPY seems to be released during high frequency neuronal activity, leading to decreased glutamate release in excitatory synapses. Importantly, memory consolidation appears to be affected by the treatment. We found that long-term potentiation (LTP) in the CA1 area is partially impaired and animals have a slower rate of hippocampal-based spatial discrimination learning. These data provide the first evidence that rAAV-based gene therapy using NPY exerts relative limited effect on synaptic plasticity and learning in the hippocampus, and therefore this approach could be considered as a viable alternative for epilepsy treatment.