Apolipoprotein ε allele 4 (APOE4) influences the metabolism of polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The entorhinal cortex (EC) in the brain is affected early in Alzheimer's disease and is rich in DHA. The purpose of this study is to identify the effect ofAPOE4and DHA lipid species on the EC. Plasma and cerebrospinal fluid (CSF) lipidomic measurements were obtained from the DHA Brain Delivery Pilot, a randomized clinical trial of DHA supplementation (n = 10) versus placebo (n = 12) for six months in nondemented older adults stratified byAPOE4status. Wild-type C57B6/J mice were fed a high or low DHA diet for 6 months followed by plasma and brain lipidomic analysis. Levels of phosphatidylcholine DHA (PC 38:6) and cholesterol ester DHA (CE 22:6) had the largest increases in CSF following supplementation (P< 0.001). DHA within triglyceride (TG) lipids in CSF strongly correlated with corresponding plasma TG lipids, and differed byAPOE4, with carriers having a lower increase than noncarriers. Changes in plasma PC DHA had the strongest association with changes in EC thickness in millimeters, independent ofAPOE4status (P= 0.007). In mice, a high DHA diet increased PUFAs within brain lipids. Our findings demonstrate an exchange of DHA at the CSF-blood barrier and into the brain within all lipid species withAPOEhaving the strongest effect on DHA-containing TGs. The correlation of PC DHA with EC suggests a functional consequence of DHA accretion in high density lipoprotein for the brain.