BACKGROUND: Previous studies have shown that primary motor cortex (M1) excitability is modulated by motor skill learning and that the M1 plays a crucial role in motor memory. However, the following questions remain: (1) At what stage do changes in M1 excitability occur? (2) Are learning-induced changes in leg M1 excitability associated with motor memory? Here, we did two experiments to answer these questions. METHODS AND RESULTS: In experiment 1, subjects learned a visuomotor tracking task over two consecutive days. Before and after the task in Day 1, we recorded input-output curves of the motor evoked potentials (I-O curve) produced in the tibialis anterior muscle by transcranial magnetic stimulation. We found that the changes in M1 excitability were affected by learning stage. In addition, the changes in M1 excitability in Day 1 were correlated with the retention. In experiment 2, we recorded I-O curves before learning, after the fast-learning stage, and after learning. We found no changes in M1 excitability immediately after the fast-learning stage. Furthermore, a significant relationship between the length of slow-learning stage and the changes in M1 excitability was detected. CONCLUSIONS: Previous studies have suggested that optimal motor commands are repeatedly used during the slow-learning stage. Therefore, present results indicate that changes in M1 excitability occur during the slow-learning stage and that such changes are proportional to motor skill retention because use-dependent plasticity occur by repetitive use of same motor commands during the slow-learning stage.