Mirror therapy (MT) is an effective approach in stroke recovery, but its impact on subcortical neural reorganization remains unclear. Thus, we aimed to investigate the neuroplastic effects on white matter due to MT. In this study, thirty-three participants with stroke were recruited and randomly assigned into the MT group (n=16) or the control group (n=17) for a 4-week intervention. Before and after the intervention, motor recovery was evaluated using the Fugl-Meyer Assessment upper limb subscale (FMA-UL), and the white matter structure and function were investigated using DTI and resting-state fMRI, focusing on the corticospinal tract and the corpus callosum. Significant correlations between the improvements of the FMA-UL and the baseline fractional anisotropy of ipsilesional corticospinal tract (p <
0.001) and corpus callosum (p = 0.009) were observed only in the MT group. Additionally, no significant structural alterations were found between the two groups after the intervention. The fractional amplitude of low-frequency fluctuation of ipsilesional corticospinal tract (p = 0.003) and corpus callosum (p = 0.005) were significantly enhanced only in the MT group, which were correlated with the improvements of the FMA-UL (p <
0.001). Furthermore, partial correlation analysis and subsequent mediation model analysis suggested that the changes of fractional amplitude of low-frequency fluctuation in corpus callosum partially mediated the effect of the baseline fractional anisotropy of ipsilesional corticospinal tract on the FMA-UL improvements in the MT group. This study provided neuroimaging evidence on white matter reorganization after MT, specifically the corpus callosum, suggesting a potential interhemispheric transcallosal neuroplastic mechanism of MT.