Background: Apolipoprotein E (ApoE) is known for its role in lipid trafficking and the e4 allele is a risk factor for late onset Alzheimers disease (AD). Recently, aberrant ceramide and fatty acid (FA) levels have been implicated in AD. Objective: To determine the specific effects of human ApoE4 (hE4) on cerebral ceramide and FA content during chow or a high fat/high cholesterol (HFHC) diet. Methods: Cerebral ceramide and FA profiles were determined by LC-MSMS in 15-month-old female wild-type (WT), ApoE-knockout (E0), and hE4-knockin mice fed chow or a HFHC diet for 3 months. mRNA levels of genes involved in ceramide and FA metabolism were determined by qPCR. Results: Similar to E0, hE4 mice displayed lower cerebral total ceramide, Cer16 : 0, and Cer24 : 1 levels than WT mice on both diets. Akin to WT mice, hE4 mice had lower total and saturated FA levels on chow than E0 mice. The HFHC diet significantly increased total and saturated FA levels in hE4 mice. Chow-fed hE4 mice showed lower mRNA levels of ceramide synthase (CerS) 6, acid sphingomyelinase, and of most ceramide and FA transporters than WT and E0 mice. The HFHC diet downregulated the expression of CerSs in hE4 and WT mice, and of ceramide and FA transporters in WT mice, but not in E0 mice. Conclusion: hE4 reduced cerebral ceramide levels to levels observed in E0 mice independent of diet. The HFHC diet increased cerebral FA levels in hE4 mice. This was associated with alterations in the expression of ceramide and FA transporters specifically in hE4 mice.