Introduction <p>Psilocybin, a classic serotonergic psychedelic, has antidepressant, anxiolytic, anti-inflammatory, and analgesic properties. However, the immunomodulatory effects of psilocybin within the central nervous system, particularly on microglial activation, remain poorly understood. Therefore, this study aimed to investigate the effects of psilocybin on microglial activation markers and cytokine levels in the spinal cord isolated from rats subjected to chronic mild stress (CMS).</p> Methods <p>Tissues were isolated from four groups of adult male Wistar Han rats, each consisting of seven animals: not subjected to CMS (control), receiving two injections of saline (0.9% NaCl <i>ip</i>) or psilocybin (0.6&#xa0;mg/kg <i>ip</i>) at 7-day intervals, and subjected to CMS, receiving two injections of NaCl (0.9% NaCl <i>ip</i>) or psilocybin (0.6&#xa0;mg/kg <i>ip</i>) at 7-day intervals. Spinal cords were collected 1 week after the second dose of saline/psilocybin from the sacrificed rats and stored at -80&#xa0;°C. The levels of microglial activation markers (iNOS and Arg1) and pro- and anti-inflammatory cytokines (TNF-α and IL-10) were analyzed using an enzyme-linked immunosorbent assay.</p> Results <p>The spinal cords isolated from CMS rats treated with psilocybin revealed significantly increased levels of Arg1 (<i>p =</i> 0.0306), TNF-α (<i>p =</i> 0.0357), and IL-10 (<i>p =</i> 0. 0081) compared to tissues from control rats administered with psilocybin. Additionally, although not statistically significant, iNOS levels showed an increasing trend in CMS rats treated with psilocybin compared to those in psilocybin-receiving controls (<i>p =</i> 0.0755).</p> Conclusions <p>CMS activates the immune system/microglia non-specifically despite psilocybin administration.</p> Clinical trial number <p>Not applicable.</p>