Introduction: : It seems that Valeriana officinalis (valerian) extract through gamma-amino-butyric acid A (GABAA) receptor possesses analgesic effect. The aim of the present study was to investigate the effect of muscimol and picrotoxin on pain sensitivity in male rats pretreated with valerian extract using the formalin test. Methods: Thirty-five male rats weighing 200-250g in standard temperature 20±2 °C and light cycle of 12/12h used. Animals were randomly divided to 7 groups: sham 1 (injection of saline); sham 2 (pretreated with valerian + ICV injection of artificial cerebro spinal fluid); experimental1 (injection of valerian extract); experimental 2 or 3 (pretreated with valerian extract + ICV injection of muscimol 250 and 500 ng/rat); experimental 4 or 5 (pretreated with valerian extract + ICV injection of picrotoxin 250 and 500 ng/rat). Valerian extract 400 mg/kg was administrated by intraperitoneal injection. Pain evaluation was done by the formalin test. Lateral ventricles cannulated unilaterally by the stereotaxic procedure. Results: Data showed that valerian extract significantly decreased pain sensitivity in the late phase of the formalin test in comparison to sham 1 group. Muscimol in both doses significantly decreased pain in comparison to sham 1, while at the dose of 500 ng/rat significantly increased pain sensitivity in comparison to sham 2 at late phases of formalin test. Picrotoxin at both doses significantly decreased pain sensitivity in comparison to sham 1, while significantly increased pain sensitivity in comparison to the sham 2 at late phases of formalin test. Conclusion: According to present results, valerian extract had analgesic effect through the GABAA receptor.