Background: The corpus callosum (CC), which connects homologous regions of the cortex, is the major conduit for information transfer between the cerebral hemispheres. Previous evidence suggested that both CC degeneration and alternations of homotopic inter-hemispheric functional connectivity are present in Alzheimer’s disease (AD) (1, 2). However, the associations between region-specific CC degeneration and homotopic inter-hemispheric functional connectivity and their relationships with memory deficits during the prodromal stage of AD remain largely unknown. We hypothesized that selective structural degeneration in CC is associated with memory impairment in AD and its prodromal stage, amnestic mild cognitive impairment (aMCI), which is mediated by homotopic inter-hemispheric functional dysconnectivity. Methods: Using structural MRI and task-free functional MRI, we examined the CC volume and inter-hemispheric functional connectivity in 66 healthy controls, 41 aMCI and 41 AD patients. Each participant underwent extensive clinical and neuropsychological evaluation, including the Clinical Dementia Rating Scale (CDR), the Mini-Mental State Examination (MMSE), the Montreal Cognitive Assessment (MoCA) and a standard neuropsychological battery (see details in our previous work). Ethics approval was obtained from the SingHealth Institutional Review Board and the National Healthcare Group Domain-Specific Review Board. The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained prior to recruitment. Results: AD patients had CC degeneration and attenuated inter-hemispheric homotopic functional connectivity. Nevertheless, aMCI patients had relatively less severe CC degeneration (mainly in the mid-anterior, central, and mid-posterior CC, sparing the anterior and posterior parts) and no reduction in inter-hemispheric homotopic functional connectivity. The degeneration of each CC sub-region was associated with specific inter-hemispheric homotopic functional disconnections in patients with AD and aMCI. More importantly, impairment of inter-hemispheric homotopic functional connectivity partially mediated the association between CC (particularly the central and posterior parts) degeneration and memory deficit. Conclusions: To our knowledge, this is the first study to demonstrate associations between region-specific CC degeneration and homotopic inter-hemispheric functional dysconnectivity, as well as their relationships with memory deficits in AD and aMCI. All CC subregions had degeneration in AD while only mid-anterior, central and mid-posterior parts exhibited shrinkage in aMCI. Homotopic inter-hemispheric functional connectivity disruption was detected in AD but not aMCI, suggesting possible compensatory mechanism. Intriguingly, impairment of inter-hemispheric homotopic functional connectivity partially mediated the associations between CC degeneration and memory deficits. These findings provide novel insights on the impact of brain inter-hemispheric structural and functional changes on memory impairment in early stage of AD.