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Objectives: To investigate the effects of paeoniflorin (PEF) on the hypothalamic-pituitary-adrenal (HPA) axis feedback function of post-traumatic stress disorder (PTSD)., Materials and Methods: Single-prolonged stress (SPS) was used to establish a PTSD-like rat model. The contents of plasma corticosterone (CORT), adrenocorticotropin hormone (ACTH) and corticotropin-releasing hormone (CRH) were measured by ELISA. Glucocorticoid receptor (GR), mineralocorticoid receptor (MR), adrenocorticotropic hormone-releasing factor I receptor (CRF1R), and adrenocorticotropic hormone-releasing factor II receptor (CRF2R) in the hippocampus and amygdala were measured by RT-PCR and immunohistochemistry., Results: The results showed that on day 8 after SPS, model rats showed enhanced HPA axis negative feedback lasting to day 29. On day 29, plasma CORT levels increased in model rats, while plasma CRH levels had no significant difference on days 8, 22, and 29. The expression of GR and MR of model rats significantly increased in the hippocampus, while the expression of GR, MR, and CRF1R significantly decreased in the amygdala. After 14 days of continuous administration of PEF, the enhanced negative feedback was inhibited, and the plasma CORT level significantly reduced after 21 days of administration. Moreover, PEF could significantly decrease the expression of GR and MR in the hippocampus, and increase the expression of GR, MR, and CRF1R significantly in the amygdala., Conclusion: PEF could regulate HPA axis dysfunction in a rat model of PTSD, which may be related to regulating expression of GR and MR in the hippocampus and amygdala and regulating expression of CRF1R in the amygdala.