The degree of involvement of the right and left hemispheres in the system matching perception and execution of actions remains unclear. Some studies have proposed that action observation involves a bilateral network of brain areas, whereas others have suggested left-hemisphere dominance. We explored hemispheric lateralization associated with action observation using transcranial magnetic stimulation over primary motor cortex devoid of callosal influences. We assessed corticospinal excitability related to observation of finger movements in one split-brain patient and six controls. Results revealed that the left hemisphere of the split-brain patient shows enhanced excitability during action observation, whereas no enhancement was found in the right hemisphere. The controls showed increased excitability in both hemispheres during action observation. Our findings suggest a left-hemisphere dominance for mirror-neuron function in the absence of a corpus callosum.