Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is highly abundant in the brain and confers protection against numerous neurological diseases, yet the fundamental mechanisms regulating the enrichment of DHA in the brain remain unknown. Here, we have discovered that a member of the long-chain acyl-CoA synthetase family, Acsl6, is required for the enrichment of DHA in the brain by generating an Acsl6-deficient mouse (Acsl6^−/−). Acsl6 is highly enriched in the brain and lipid profiling of Acsl6^−/− tissues reveals consistent reductions in DHA-containing lipids in tissues highly abundant with Acsl6. Acsl6^−/− mice demonstrate motor impairments, altered glutamate metabolism, and increased astrogliosis and microglia activation. In response to a neuroinflammatory lipopolysaccharide injection, Acsl6^−/− brains show similar increases in molecular and pathological indices of astrogliosis compared with controls. These data demonstrate that Acsl6 is a key mediator of neuroprotective DHA enrichment in the brain.