Ethnopharmacology relevance: Post-traumatic stress disorder (PTSD) is a severe psychiatric disorder that is characterized by symptoms of re-experiencing, avoidance and hyperarousal, as well as social and professional dysfunction at least one month after the exposure to a traumatic event. Biosynthesis of allopregnanolone has been suggested as one of the important contributors to PTSD. Albiflorin (AF) extracted from Radix paeoniae Alba had been shown to be effective in the therapy of depression. However, few studies were concerned about the anti-PTSD-like effects of AF. Aim of the study: The current study aimed to evaluate the anti-PTSD-like effects of AF in an animal model and its possible mechanism Materials and methods: To evaluate this, the single prolonged stress (SPS) model was used in the present study. The SPS rats were administered by AF (at doses of 3.5, 7 and 14.0 mg/kg, kg.) after induction of SPS from days 2-13. After the exposure to SPS, behavioral assessments were conducted, including contextual fear paradigm (CFP), elevated plus-maze test (EPMT), open-field test (OFT). The rats were decapitated at the end of the behavioral tests and levels of allopregnanolone in prefrontal cortex, hippocampus and amygdala were measured by enzyme linked immunosorbent assay (ELISA). Results: It had been shown that behavioral deficits of SPS rats were reversed by AF (7.0 and 14.0 mg/kg, i.g.), which attenuated the PTSD-like associated contextual freezing behavior in CFP and improved PTSD-like associated anxiogenic behavior in EPMT without affecting locomotor activity in OFT. Moreover, decreased levels of allopregnanolone in prefrontal cortex, hippocampus, and amygdala were reversed by AF (7.0 and 14.0 mg/kg, i.g.), respectively. Conclusion: In summary, the present study indicated that AF exerted the anti-PTSD-like effects, which maybe associated with allopregnanolone biosynthesis in the brain.