Background <p><?tk 1?>Post-traumatic stress disorder (PTSD) is associated with dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis and the sympatho-adrenomedullary system, leading to immune imbalance and alterations in microbiota composition. Stress-induced disruption of intestinal barrier integrity may promote bacterial translocation to peripheral organs, including the spleen. Aripiprazole (ARI), an atypical antipsychotic proposed for PTSD treatment, also modulates the immune system and microbiota. This study investigated the effects of ARI on splenic microbiota composition and splenic neuroendocrine and immune responses in an animal model of PTSD.</p> Methods <p><?tk 1?>Rats were exposed to the single prolonged stress (SPS) paradigm to induce a PTSD-like phenotype and treated intraperitoneally with vehicle or ARI for 28 days. Anxiety-like behavior was assessed using the elevated plus maze. Splenic microbiota and gene expression were quantified by real-time PCR in isolated splenocytes following ex vivo stimulation with lipopolysaccharide or phorbol 12-myristate 13-acetate (PMA)/ionomycin.</p> Results <p><?tk 1?>SPS and SPS + ARI reduced the abundance of the phylum <i>Bacteroidetes</i>. SPS increased <i>γ/δ-Proteobacteria</i> and <i>Lactobacillus</i> abundance, effects attenuated by ARI. The presence of specific splenic bacteria correlated with anxiety-like behavior. While lipopolysaccharide-induced responses were unaffected, splenocytes from SPS-exposed rats exhibited increased expression of Th1- and Th17-related genes after PMA/ionomycin stimulation; this effect was reversed by ARI.</p> Conclusion <p><?tk 1?>ARI modulates SPS-induced alterations in splenic microbiota and attenuates heightened Th1- and Th17-associated responses, thereby contributing to the restoration of immune balance. Our findings underscore involvement of the gut–spleen–brain axis in PTSD pathogenesis and immunosuppressive/ microbiota-modulating effects of aripiprazole on splenic immune cells.</p> Graphical Abstract <p></p>