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Purpose: To investigate the potency of ampicillin in altering gut flora in the presence of a high-sucrose diet in rat pups, and to determine its effect on selected neurotransmitters and a cytokine as markers of the persistent autistic features repeatedly induced in orally administered propionic acid rat pups.. Methods: Twenty-eight young male Wistar albino rats were divided into four equal groups. The first group served as a control. The second group received an oral neurotoxic dose of propionic acid (PPA, 250 mg/kg body weight/day) for 3 days. The third group was treated with ampicillin (50 mg/kg for 3 weeks) with a standard diet. The fourth group was given the same dose of ampicillin with a high-sucrose diet for 10 weeks. Results: The results showed a significant ( p < 0.001) decrease in the investigated neurotransmitters in PPA- and ampicillin-treated rat pups (norepinephrine by 32.49 and 14.58 %, dopamine by 31.45 and 20.22 %, serotonin by 35.99 and 29.09 %), as well as a remarkable increase ( p < 0.001) in the proinflammatory cytokine, IL-6 (30.07and 6.07 %). The high-sucrose diet also significantly ( p < 0.001) enhanced the neurotoxic effect of ampicillin. Conclusion: The observed dietary modulation of the gut microbiota, coupled with the subsequent modulation of brain neurochemistry and inflammation, demonstrates the considerable potential of dietary intervention through the elimination of highly refined sugar as a treatment strategy to prevent and treat autism. Keywords: Neurotoxicity, Ampicillin, Propionic acid, Neurotransmitters, Cytokines, High-sucrose diet