Childhood adrenoleukodystrophy (cALD) is a metabolic disorder in which very long-chain fatty acids (VLCFA) accumulate due to ALD protein gene defects, ultimately leading to lipotoxicity-induced neuroinflammatory demyelinating disease. Therefore, we examined VLCFA-mediated alterations in the metabolism of lipoxidative enzymes and inflammatory mediators in the cALD brain. 5-Lipoxygenase (5-LOX)-derived leukotrienes were significantly elevated in all the areas of white matter in the cALD brain. Unlike cyclooxygenase-2 expression, which was moderately high only in the plaque area, expression of 5-LOX and cytosolic phospholipase A2 was prominent in all the areas. This lipoxidative burden in the cALD brain was further shown by reduced levels of glutathione and enhanced expression of heat shock protein-70/manganese superoxide dismutase. These pathological observations were confirmed through in vitro mechanistic investigation. After increasing VLCFA through silencing Abcd1+Abcd2 in mouse primary astrocytes, enhanced expression of 5-LOX was observed, and this increased expression was blocked by treatment with monoenoic fatty acids. These results link the previously observed accumulation of VLCFA in cALD to the 5-LOX enzyme pathway. A similar increase in 5-LOX expression in astrocytes was also detected following treatment with exogenous VLCFA (C26:0). In sum, through 5-LOX activation, VLCFA accumulation causes a lipotoxic response consistent with cALD brain pathology.