Objective This study aimed to investigate the morphometric differences in the corpus callosum between patients with major depressive disorder (MDD) and healthy controls and analyze their relationship to gray matter changes. Methods Twenty female MDD patients and 21 healthy controls (HCs) were included in the study. To identify the difference in the regional gray matter concentration (GMC), VBM was performed with T1 magnetic resonance imaging. The shape analysis of the corpus callosum was processed. Diffusion tensor imaging (DTI) fiber-tracking was performed to identify the regional tract pathways in the damaged corpus callosal areas. Results In the shape analysis, regional shape contractions in the rostrum and splenium were found in the MDD patients. VBM analysis showed a significantly lower white matter concentration in the genu and splenium, and a significantly lower GMC in the frontal, limbic, insular, and temporal regions of the MDD patients compared to the HCs. In DTI fiber-tracking, the fibers crossing the damaged areas of the genu, rostrum, and splenium were anatomically connected to the areas of lower GMC in MDD patients. Conclusion These findings support that major depressive disorder may be due to disturbances in multiple neuronal circuits, especially those associated with the corpus callosum.