We previously reported that repetitive paired-pulse transcranial magnetic stimulation (TMS
rPPS) synchronized to the peak phase of transcranial alternating current stimulation (tACS) at the β frequency induced long-lasting after-effects on primary motor cortex (M1) with less inter-individual variability compared with rPPS alone. Here, we investigated the plasticity mechanisms underlying combined stimulation effects using paired-pulse TMS paradigms. rPPS was applied to the peak phase of β tACS (rPPS-tACS-peak) or sham tACS (rPPS alone), or tACS was delivered without rPPS (tACS alone). Resting motor threshold (RMT) and motor evoked potentials (MEPs) elicited by single-pulse TMS, short-interval intracortical inhibition (SICI), intracortical facilitation (ICF), short-latency afferent inhibition (SAI), and short-interval intracortical facilitation (SICF) were measured before and after intervention. rPPS-tACS-peak stimulation significantly increased MEPs compared with other conditions after intervention. Although I-wave interaction was expected to be produced by the facilitation effect of rPPS, rPPS-tACS-peak did not change SICF. In contrast, SAI was decreased in rPPS-tACS-peak compared with baseline. In the control experiment, rPPS-tACS-trough did not change MEPs, SAI, and SICF. Therefore, the after-effects of rPPS-tACS-peak on M1 may be caused by a partial reduction in the inhibitory circuit mediated by cholinergic interneurons, rather than an enhancement of the facilitatory effects of rPPS.