Despite apolipoprotein E's important role in cholesterol transport and metabolism in the brain as well as its influence on Alzheimer's disease, the impact of the human APOE genotype on cholesterol metabolism in brain has not been fully examined. This study was carried out to investigate APOE genotype effects on oxysterols measured. In this study the measurement of cholesterol and several oxysterols in the brains of human APOE epsilon2, epsilon3 and epsilon4 knock-in mice at 8 weeks and 1 year of age using gas chromatography mass spectrometry (GC-MS) demonstrated no APOE genotype or age effect on total brain cholesterol and the oxysterol 24-hydroxycholesterol. The level of 27-hydroxycholesterol was elevated in 1 year old animals for all APOE genotypes. Interestingly, lathosterol an indicator of cholesterol synthesis was significantly reduced in the 1 year old animals for all APOE genotypes. APOE epsilon4 expressing mice exhibited statistically lower levels of lathosterol compared to APOE epsilon2 in both the young and old mice. Oxidized cholesterol metabolites were significantly lower in APOE epsilon2 mice compared to other genotypes at 8 weeks old. Although minimal differences were observed between APOE E3 and E4 knock-in (KI) mice, these findings indicate that there are some clear APOE genotype specific effects on brain cholesterol synthesis and associated metabolic pathways, particularly in APOE epsilon2 KI mice.