Long-chain polyunsaturated fatty acids (LCPUFA) which are synthesized mainly in the liver have relevant functions in the organism.A diet high in fat (HFD) generates an increase in the levels of fat and induces oxidative stress (lipo-peroxidation) in the liver, along with a reduction in tissue n-3 and n-6 LCPUFA.Extra virgin olive oil (EVOO) is rich in anti-oxidants (polyphenols and tocopherols) which help to prevent the development of oxidative stress.This study evaluated the role of EVOO in preventing the induction of fat deposition and oxidative stress in the liver and in the depletion of LCPUFA in the liver, erythrocytes and brain generated by a HFD in C57BL/6J mice.Four experimental groups (n = 10/group) were fed a control diet (CD) or a HFD for 12 weeks and were respectively supplemented with EVOO (100 mg/day).The group fed HFD showed a significant increase (p < 0.05) in fat accumulation and oxidative stress in the liver, accompanied by a reduction in the levels of n-3 and n-6 LCPUFA in the liver, erythrocytes and brain.Supplementation with EVOO mitigated the increase in fat and oxidative stress produced by HFD in the liver, along with a normalization of LCPUFA levels in the liver, erythrocytes and brain.It is proposed that EVOO supplementation protects against fat accumulation, and oxidative stress and normalizes n-3 and n-6 LCPUFA depletion induced in mice fed a HFD.