Synaptic plasticity plays a crucial role in learning, memory, and cognitive disorders. Cytoskeletal reorganization underlies neuronal synaptic plasticity, but little is known about the regulation of cytoskeletal dynamics in living animals. We used stable isotope labeling to measure the turnover of tubulin in defined microtubule (MT) populations in murine brain. Neuronal MTs generally exhibited low turnover rates in vivo. Basal turnover was highest in tau-associated MTs, intermediate in microtubule-associated protein 2 (MAP2)–associated MTs, and lowest in cold-stable MTs. Labeling of MTs in mature neurons in cell culture yielded similar turnover results. Intracerebroventricular glutamate injection stimulated, via N-methyl-d-aspartic acid receptors, label incorporation (turnover) in cold-stable, tau-associated, and MAP2-associated MTs, the last of which was shown to be dependent on cyclic adenosine-3′, 5′-monophosphorothioate–protein kinase A. Contextual fear conditioning, a hippocampus-mediated form of memory formation, was accompanied by increased turnover of hippocampal MAP2-associated and cold-stable MTs. Treatment with the MT-depolymerizing drug nocodazole reversed the conditioning-induced increase in label incorporation in MAP2-associated MTs, reduced dendritic spine density, and impaired memory formation. The effects of nocodazole on MT turnover were prevented by the MT-stabilizing agent Taxol (Sigma-Aldrich, St. Louis, MO, USA) and by brain-derived nerve growth factor, both of which also restored dendritic spine density and memory formation in this model. In conclusion, these results suggest that changes in hippocampal MT turnover are required for, and are a biomarker of, the synaptic plasticity that is involved in memory formation.