Background: Glycosylation is an important post-translational modification of apolipoprotein E (APOE) and APOE in cerebrospinal fluid (CSF) displays an additional secondary glycosylation that is not present in plasma, suggesting a brain-specific regulatory mechanism. This study investigated whether altered APOE glycosylation is associated with early cognitive decline and progression from mild cognitive impairment (MCI) to AD and further explored its potential biological relevance. Methods: APOE glycosylation levels were measured in participants with MCI and healthy controls (HCs). Analyses were stratified by APOE4 genotype and examined in relation to neuroimaging markers. The data-derived cut-off values were used to assess disease progression. Single-cell transcriptomic data from the SEA-AD cohort were analyzed to characterize glycosylation-related alterations in dementia microglia. Results: The total APOE glycosylation in CSF and plasma did not differ significantly between the MCI and control groups. CSF secondary APOE glycosylation was lower in participants with MCI in the unadjusted analysis. It was not significantly associated with baseline white matter hyperintensity (WMH) or hippocampal volume. In exploratory analyses using a data-derived cut-off, lower CSF secondary glycosylation was associated with faster hippocampal volume decline and shorter time to AD conversion. Donor-level pseudobulk analysis of the SEA-AD cohort showed lower ST6GAL1 expression and higher APOE, LPL, and CD9 expression in donors with dementia; MGAT1 was not significantly altered overall. Conclusions: CSF secondary APOE glycosylation may be associated with early pathological changes and disease progression in MCI. Together with alterations in microglial glycosylation-related pathways, these exploratory findings suggest broader glycosylation dysregulation in APOE-related neurodegeneration.