BackgroundTemporal lobe epilepsy (TLE), a prevalent neurological disorder, is associated with hippocampal oxidative stress and inflammation. A recent study reveals that the long noncoding RNA ILF3 divergent transcript (ILF3‐AS1) level is elevated in the hippocampus of TLE patients; however, the functional roles of ILF3‐AS1 in TLE and underlying mechanisms deserve further investigation. Hence, this study aimed to elucidate whether ILF3‐AS1 is involved in the pathogenesis of TLE by regulating oxidative stress and inflammation and to explore its underlying mechanism in vitro.MethodsHuman hippocampal neurons were subjected to a magnesium‐free (Mg2+‐free) solution to establish an in vitro model of TLE. The potential binding sites between ILF3‐AS1 and miRNA were predicted by TargetScan/Starbase and confirmed by dual luciferase reporter assay. Cell viability and damage were assessed by cell counting kit‐8 and lactate dehydrogenase assay kits, respectively. Levels of reactive oxygen species, malondialdehyde, and superoxide dismutase were determined by commercial kits. Levels of Interleukin‐6 (IL‐6), IL‐1β, and tumor necrosis factor‐alpha were quantified by enzyme‐linked immunosorbent assay. The expressions of gene and protein were determined by quantitative real‐time polymerase chain reaction and Western blot analysis.ResultsIn Mg2+‐free‐treated hippocampal neurons, both ILF3‐AS1 and HMGB1 were highly up‐regulated, whereas miR‐504‐3p was down‐regulated. ILF3‐AS1 knockdown ameliorated Mg2+‐free‐induced cellular damage, oxidative stress, and inflammatory response. Bioinformatics analysis revealed that miR‐504‐3p was a target of ILF3‐AS1 and was negatively regulated by ILF3‐AS1. MiR‐504‐3p inhibitor blocked the protection of ILF3‐AS1 knockdown against Mg2+‐free‐induced neuronal injury. Further analysis presented that ILF3‐AS1 regulated HMGB1 expression by sponging miR‐504‐3p. Moreover, HMGB1 overexpression reversed the protective functions of ILF3‐AS1 knockdown.ConclusionOur findings indicate that ILF3‐AS1 contributes to Mg2+‐free‐induced hippocampal neuron injuries, oxidative stress, and inflammation by targeting the miR‐504‐3p/HMGB1 axis. These results provide a novel mechanistic understanding of ILF3‐AS1 in TLE and suggest potential therapeutic targets for the treatment of epilepsy.