The primary motor cortex (M1) is fundamentally important for the acquisition of skilled motor behaviours. Recent advances in imaging and electrophysiological techniques have improved our understanding of M1 neural circuit modulation in rodents and non-human primates during motor learning. However, little remains known about the learning-related changes of distinct elements in the human brain. In this study, we tested excitability changes of different neural circuits (infragranular and supragranular layers) in the M1 of human subjects who underwent training in a discrete spatiotemporal motor skill. Excitability modulations were assessed by recording H-reflex facilitation from transcranial magnetic stimulation at movement onset. Motor practice improved the consistency of movements and was accompanied by an excitability increase of the fastest corticospinal connections during the initial stages of motor practice. No such excitability changes were observed for training in a simple motor skill and circuits at supragranular layers of M1. Notably, changes in excitability were not associated with changes in motor performance. Our findings could reflect learning-related increases in the recruitment and/or reorganisation of the fastest corticospinal connections.