To assess the effect of Rhoifolin (ROF [apigenin 7-O-enneohesperidoside]) on the damage to hippocampal neu-ronal culture model of acquired epilepsy (AE) and investigate its possible mechanisms. A hippocampal neuronal culture model of AE was established through incubating HT-22 cells with MgCl2 free medium. 3-(4,5-Dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to assess the effect of ROF on cell viability and apoptosis exposed to epilepsy. The oxidative stress and secretion of inflammatory cytokines were measured by reverse transcription-quantitative polymerase chain reaction and enzyme-linked-immunosorbent serologic assay, respectively. Immunoblot assays were performed to determine the protein expression levels of nuclear factor kappa B/nitric oxide synthases/cyclooxygenase-2 (NF-κB/iNOS/COX-2) axis. ROF increases viability and reduces apoptosis of AE medium-treated HT-22 cell line. ROF relieves oxidative stress in AE medium-treated HT-22 cell line. ROF decreases the levels of pro-inflammatory cytokines in AE medium-treated HT-22 cell line. The functional effects of ROF on AE medium-treated HT-22 cell line is through inhibiting NF-κB/iNOS/COX-2 axis. ROF increased viability, decreased apoptosis, suppressed oxidative stress, and reduced pro-inflammatory cytokine levels in an epilepsy model in vitro by inhibiting NF-κB/iNOS/COX-2 axis. ROF might serve as a potential drug for epilepsy treatment.