We postulated that gradual muscle relaxation during motor learning would dynamically change activity in the primary motor cortex (M1) and modify short-interval intracortical inhibition (SICI). Thus, we compared changes in M1 excitability both pre and post motor learning during gradual muscle relaxation. Thirteen healthy participants were asked to gradually relax their muscles from an isometric right wrist extension (30% maximum voluntary contraction; MVC) using a tracking task for motor learning. Single or paired transcranial magnetic stimulation (TMS) was applied at either 20% or 80% of the downward force output during muscle release from 30% MVC, and we compared the effects of motor learning immediately after the 1st and 10th blocks. Motor-evoked potentials (MEPs) from the extensor and flexor carpi radialis (ECR and FCR) were then measured and compared to evaluate their relationship before and after motor learning. In both muscles and each downward force output, motor cortex excitability during muscle relaxation was significantly increased following motor learning. In the ECR, the SICI in the 10th block was significantly increased during the 80% waveform decline compared to the SICI in the 1st block. In the FCR, the SICI also exhibited a greater inhibitory effect when muscle relaxation was terminated following motor learning. During motor training, acquisition of the ability to control muscle relaxation increased the SICI in both the ECR and FCR during motor termination. This finding aids in our understanding of the cortical mechanisms that underlie muscle relaxation during motor learning.