AbstractApolipoprotein E (ApoE)’s ϵ4 alle is the most important genetic risk factor for late onset Alzheimer's Disease (AD). Cell‐surface heparan sulfate (HS) is a cofactor for ApoE/LRP1 interaction and the prion‐like spread of tau pathology between cells. 3‐O‐sulfo (3‐O‐S) modification of HS has been linked to AD through its interaction with tau, and enhanced levels of 3‐O‐sulfated HS and 3‐O‐sulfotransferases in the AD brain. In this study, we characterized ApoE/HS interactions in wildtype ApoE3, AD‐linked ApoE4, and AD‐protective ApoE2 and ApoE3‐Christchurch. Glycan microarray and SPR assays revealed that all ApoE isoforms recognized 3‐O‐S. NMR titration localized ApoE/3‐O‐S binding to the vicinity of the canonical HS binding motif. In cells, the knockout of HS3ST1‐a major 3‐O sulfotransferase‐reduced cell surface binding and uptake of ApoE. 3‐O‐S is thus recognized by both tau and ApoE, suggesting that the interplay between 3‐O‐sulfated HS, tau and ApoE isoforms may modulate AD risk.