Synaptic plasticity via AMPA receptor trafficking at synapse terminals is associated with memory and learning. To investigate the neuronal mechanism of motor learning, we performed a rotor rod test and made acute brain slice to analyze layers II/III neurons in the rat motor cortex using patch clamp method. Motor skill consistently improved within 2 days of training in all animals (n = 22). In current clamp analysis, motor learning for 2 days significantly decreased firing threshold and increased firing probability, since the resting membrane potential significantly increased after motor learning. In voltage clamp analysis on the 1st day, the trained rats showed significant increase in the AMPA/NMDA ratio and miniature EPSC (mEPSC) amplitude but not frequency (ANOVA and Fisher's test, P < 0.05), suggesting that changes in the sensitivity of postsynaptic AMPA receptors are responsible for the early phase of motor learning. On the 2nd day, the AMPA/NMDA ratio increase was not observed, probably due to an increase of NMDA current. In addition, the increase of both mEPSC amplitude and frequency and the decrease of paired-pulse responses ware induced (ANOVA and Fisher's test, P < 0.05), indicating that the increase of glutamate release is involved at the late phase of learning. On the other hand, only miniature IPSC (mIPSC) frequency decreased on the 1st day. Further, motor learning produced a significant correlation in the amplitude between mEPSC and mIPSC on the 1 day and 2nd day (Spearman's correlation, P < 0.05). These results suggest the learning-induced plasticity dependent on the different leaning phase in layers II/III neurons of primary motor cortex.