Paeoniflorin (PF) is a principle active ingredient of Paeoniae Radix (PR), which has been used in Chinese traditional medicine for years, in relieving pain, especially visceral pain. The present study evaluated the antinociceptive effect of PF in neonatal maternal separation (MS) induced visceral hyperalgesia in rats, and further investigated its effective targets and the possible involvement of opioid and alpha 2-adrenergic receptors in its antinociception process. The analgesic effect of PF was evaluated by both abdominal withdraw reflex (AWR) test and abdominal electromyography (EMG) recording in response to colorectal distension (CRD, three cycles of gradient distension: 20, 40, 60, 80 mmHg, 20 seconds duration, 2 minutes interval between distension). PF (45, 90, 180, 360 mg/kg, i.p. injection) produced a dose-dependent analgesic effect in MS rats, and the maximal effect was observed 30 minute after the i.p. injection. The analgesic targets of PF might be in the central nervous system (CNS), as intracerebral ventricular (i.c.v.) administration of PF (4.5mg/kg) produced the same antinociceptive effects as the i.p. administration of PF at 45mg/kg. HPLC analysis showed that PF distributed in various brain regions, like the hypothalamus, amygdale and the frontal cortex, 30 minute after i.p. injection, and the highest PF concentration was found in the hypothalamus. Besides, immunohistochemical results showed that PF decreased fos expression induced by CRD in ventromedial hypothalamic nucleus. The analgesic effect of PF in MS rats could be partially reversed by nor-binaltorphimine, a kappa-opioid receptor antagonist and significantly reversed by DL-alpha-methyltyrosine, a catecholamines synthesis inhibitor, and yohimbine, an alpha 2-adrenoceptor antagonist.Our findings suggest that PF has antinociceptive effects in MS rats, its effects may be mediated by kappa-opioid and alpha 2-adrenergic receptors in the CNS.