Psilocybe cubensis is a species of psilocybin mushroom (magic mushroom) of moderate potency whose principal active compounds are psilocybin and psilocin. Recent studies have shown the significant procognitive and mood-enhancer effects of Psilocybe cubensis. However, evidence is so limited, especially in preclinical studies. We aimed to investigate the effect of Psilocybe cubensis extract on posttraumatic stress disorder (PTSD)-like behavior, pain perception, locomotor activity, and anxiety in a rat model of PTSD. Male rats were exposed to three consecutive shocks (0.8 mA, 3 s interval) paired with three sounds broadcasted 3 s before delivering shocks (75 dB, 3 s). After 1, 3, or 21 days, freezing rate was measured in the fear-conditioning apparatus. Open filed test and hot plate were used to assess locomotor activity and anxiety, and pain subthreshold, respectively. Psilocybe cubensis was injected intraperitoneal at the dose of 25 mg/kg (single administration) before (pretrain) or after (posttrain) shocks, or before the test (pretest). Results showed psilocybin potently alleviated PTSD symptom is short-but not long-term after the induction of PTSD. Psilocybe cubensis decreased locomotor activity only in a short period after administration. Psilocybe cubensis also increased pain subthreshold and decreased anxiety. In conclusion, Psilocybe cubensis effects on PTSD-like behavior and locomotor activity seem to be remained in short-term, while Psilocybe cubensis effects on pain subthreshold and anxiety remained long-term. This is the first study evaluating the effect of Psilocybe cubensis on PTSD-like behavior in rats in three different time protocols (1, 3, and 21 days after fear conditioning).