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The compound was administered at doses of 4, 20, and 40\u00a0mg\/kg and showed no signs of toxicity after 96\u00a0h of observation at any of the doses tested. In the open field test, withanolide D reduced locomotor activity, indicating a sedative\u2010like effect. Nevertheless, in the light\/dark test, doses of 20 and 40\u00a0mg\/kg produced an anxiolytic\u2010like response, suggesting that anxiolytic and motor effects may partially overlap depending on dose. The anxiolytic effect observed at the minimum effective dose (20\u00a0mg\/kg) was reversed by flumazenil (FMZ), supporting the involvement of a benzodiazepine\u2010sensitive GABA\n                    <jats:sub>A<\/jats:sub>\n                    modulatory domain. Furthermore, withanolide D, at a dose of 4\u00a0mg\/kg, prevented ethanol\u2010induced memory impairment in the inhibitory avoidance task, suggesting a protective effect on memory consolidation. Molecular docking analyses revealed favorable interactions of withanolide D at the extracellular \u03b11\u03b32 interface of the GABA\n                    <jats:sub>A<\/jats:sub>\n                    receptor, supporting a putative allosteric interaction in a functionally related modulatory region rather than definitive occupation of the classical diazepam site. Consistently, normal mode analysis (NMA) showed that withanolide D increases receptor mobility compared to diazepam, with RMSF values reaching up to 0.98 \u00c5, indicating enhanced structural flexibility and dynamic modulation of the protein. 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