Exercise is a potent enhancer of learning and memory, yet we know little of the underlying mechanisms that likely include alterations in synaptic efficacy in the hippocampus. To address this issue, we exposed mice to a single episode of voluntary exercise, and permanently marked activated mature hippocampal dentate granule cells using conditional Fos-TRAP mice. Exercise-activated neurons (Fos-TRAPed) showed an input-selective increase in dendritic spines and excitatory postsynaptic currents at 3 days post-exercise, indicative of exercise-induced structural plasticity. Laser-capture microdissection and RNASeq of activated neurons revealed that the most highly induced transcript was Mtss1L, a little-studied I-BAR domain-containing gene, which we hypothesized could be involved in membrane curvature and dendritic spine formation. shRNA-mediated Mtss1L knockdown in vivo prevented the exercise-induced increases in spines and excitatory postsynaptic currents. Our results link short-term effects of exercise to activity-dependent expression of Mtss1L, which we propose as a novel effector of activity-dependent rearrangement of synapses.