Background: Coactivation of primary motor cortex ipsilateral to a unilateral movement (M1ipsilateral) has been observed, and the magnitude of activation is influenced by the contracting muscles. It has been suggested that the microstructural integrity of the callosal motor fibers (CMFs) connecting M1 regions may reflect the observed response. However, the association between the structural connectivity of CMFs and functional changes in M1ipsilateral remains unclear. The purpose of this study was to investigate the relationship between functional changes within M1 ipsilateral during unilateral arm or leg movements and the microstructure of the CMFs connecting both homotopic representations (arm or leg). Methods: Transcranial magnetic stimulation was used to assess changes in motor evoked potentials (MEP) in an arm muscle during unilateral movements compared to rest in fifteen healthy adults. Functional magnetic resonance imaging was then used to identify regions of M1 associated with either arm or leg movements. Diffusion-weighted imaging data was acquired to generate CMFs for arm and leg areas using the areas of activation from the functional imaging as seed masks. Individual values of regional fractional anisotropy (FA) of arm and leg CMFs was then calculated by examining the overlap between CMFs and a standard atlas of corpus callosum. Results: The change in the MEP was significantly larger in the arm movement compared to the leg movement. Additionally, regression analysis revealed that FA in the arm CMFs was positively correlated with the change in MEP during arm movement, whereas a negative correlation was observed during the leg movement. However, there was no significant relationship between FA in the leg CMF and the change in MEP during the movements. Conclusions: These findings suggest that individual differences in interhemispheric structural connectivity may be used to explain a homologous muscle-dominant effect within M1ipsilateral hand representation during unilateral movement with topographical specificity.