Gonadal hormones such as estradiol have a powerful effect on neuronal plasticity, particularly in the hippocampus, a region crucial for memory consolidation and spatial cognition. Previous in vitro work has found that estradiol triggers second messenger cascades, driving synaptic plasticity in the hippocampus. In particular, estradiol has been found to drive a pronounced proliferation of dendritic spines, the primary sites of excitatory synaptic connections. However, the effect of the estrous cycle on neural plasticity is poorly understood due to the challenge of longitudinally tracking the structural and functional properties of hippocampal neurons in intact mice. To address this challenge, we took advantage of new approaches for longitudinal two-photon imaging in the hippocampus of behaving mice using implanted microprisms. We used this approach to measure changes in neuronal morphology, dendritic processing, and spatial coding in hippocampal neurons throughout the estrous cycle. Our results indicate that the estrous cycle profoundly modulates spatial processing, from the level of individual synaptic connections to the level of spatial coding of neuronal populations.