Inhibitory neural control of antagonist muscle is one of the fundamental neural mechanism of coordinated human limb movement. Previous studies have revealed that motor execution (ME) and motor imagery (MI) share many common neural substrates; however, whether inhibitory neural activity occurs during MI remains unknown. In addition, recent studies have demonstrated that a combined MI and action observation (MI + AO) produces strong neurophysiological changes compared with MI or AO alone. Therefore, we investigated inhibitory changes in cortical and spinal excitability of the antagonist muscle during MI + AO and ME. Single-pulse transcranial magnetic stimulation (TMS) experiments revealed that corticospinal excitability of the antagonist muscle was decreased during MI + AO. Conversely, F-wave experiments showed that F-wave persistence of the antagonist muscle increased. Paired-pulse TMS experiment also demonstrated that short-interval intracortical inhibition (SICI) did not contribute to this inhibition. Therefore, cortical mediated inhibition, except for SICI, may be related to this inhibition. Conversely, such clear inhibition of the antagonist muscle was not observed during ME, presumably owing to the effects of muscle contraction to decelerate the movements and/or sensory input accompanying the joint movements. These findings provide important insights into the neurophysiological differences between MI + AO and ME.