This study employed a test-retest design to examine changes in brain activity associated with practice of a motor skill. We recorded EEG activity from twelve right-handed recreational golfers (mean handicap: 23) as they putted 50 balls to a 2.4m distant hole, before and after a 3-day practice. We measured changes in putting performance, conscious processing, and regional EEG alpha activity. Putting performance improved and conscious processing decreased after practice. Mediation analyses revealed that performance improvements were associated with changes in EEG alpha, whereby activity in task-irrelevant cortical regions (temporal regions) was inhibited and functionally isolated from activity in task-relevant regions (central regions). These findings provide evidence for the development of greater neurophysiological efficiency with practice of a motor skill.