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Cortical reorganization within the primary motor cortex (M1) contralateral to a practicing hand has been extensively investigated. The extent to which the ipsilateral M1 participates in these plastic changes is not known. Here, we evaluated the influence of unilateral hand practice on the organization of the M1 ipsilateral and contralateral to the practicing hand in healthy human subjects. Index finger movements elicited by single-pulse transcranial magnetic stimulation (TMS) delivered to each M1 were evaluated before and after practice of unilateral voluntary index finger abduction motions. Practice increased the proportion and acceleration of TMS-evoked movements in the trained direction and the amplitude of motor-evoked potentials (MEPs) in the abduction agonist first dorsal interosseous (FDI) muscle in the practicing hand and decreased the proportion and acceleration of TMS-evoked abduction movements and MEP amplitudes in the abduction agonist FDI in the opposite resting hand. Our findings indicate that unilateral hand practice specifically weakened the representation of the practiced movement in the ipsilateral M1 to an extent proportional to the strengthening effect in the contralateral M1, a result that varied with the practicing hand's position. These results suggest a more prominent involvement of interacting bilateral motor networks in motor memory formation and probably acquisition of unimanual motor skills than previously thought.