It has long been postulated that apolipoprotein E (apoE) may play a role in lipid metabolism in the brain. However, direct evidence that apoE plays such a role is lacking. We investigated whether apoE isoforms influence lipid content in the brain. We compared the brains of wild-type mice to apoE knockout (-/-) and human apoE3 and apoE4 transgenic mice and compared cerebrospinal fluid (CSF) of humans with different apoE isoforms. We found that there was no effect of apoE on the content of multiple phospholipids, sphingolipids, and cholesterol. There was, however, a marked effect of apoE on the sulfatide (ST) content in both the brain and CSF. The sulfatide mass in hippocampus and cortex of apoE knockout mice was found to be 61 and 114 mol% higher than wild-type mice counterparts at 12 months of age. In contrast, the sulfatide content in brain tissues from human apoE4-expressing mice was ∼60% less than those found in wild-type mice of the same age. The ST mass in human CSF was significantly dependent on the APOE genotypes of the subjects. Examination of potential sulfatide carrier(s) in human CSF demonstrated that sulfatides are specifically associated with apoE-containing high density lipoproteins, suggesting that sulfatide levels in the central nervous system (CNS) are likely to be directly modulated by the same metabolic pathways that regulate levels of apoE-containing CNS lipoproteins. This novel role for apoE in the CNS may provide new insights into the connection of apoE with Alzheimer's disease and poor recovery after brain injury.