Inhibition of estrogen synthesis by aromatase inhibitors has become a favoured therapy for breast cancer in postmenopausal women. Estrogen is, however, important for synapse formation in the hippocampus. Inhibition of aromatase induces spine synapse loss in organotypic hippocampal slice cultures. We therefore studied the effect of systemic treatment with the potent aromatase inhibitor letrozole, for periods of seven days and four weeks, on spine formation and synaptic proteins in the hippocampi of female mice. In cyclic, letrozole-treated females and in ovariectomized, letrozole-treated females, the number of spine synapses was significantly reduced in the hippocampus but not in the prefrontal or cerebellar cortex. Consequently, the expression of NMDA receptors NR1 and NR2b were down regulated after treatment with letrozole and long-term potentiation (LTP) was dramatically impaired. In cyclic animals the expression of the synaptic proteins synaptophysin and spinophilin was down regulated in response to letrozole. In ovariectomized animals, however, protein expression was down regulated after seven days of treatment, whereas the expression was up regulated after four weeks of treatment. Our results indicate that systemic inhibition of aromatase in mice affects structural synaptic plasticity and LTP in the hippocampus. This may be an underlying cause of cognitive deficits in postmenopausal women treated with aromatase inhibitors.