This experiment was performed to investigate whether obovatol isolated from the leaves of Magnolia obovata has anxiolytic-like effects through GABA-benzodiazepine-receptors Cl− channel activation. The anxiolytic-like effects of obovatol in mice were examined using the elevated plus-maze and the automatic hole-board apparatus. Oral administration of obovatol (0.2, 0.5 and 1.0 mg/kg) significantly increased the number of open arm entries and the spent time on open arm in the elevated plus-maze test, compared with those of saline. Obovatol (0.2, 0.5 and 1.0 mg/kg) also produced anxiolytic-like effects, as reflected by an increase in head-dipping behaviors. These effects were comparable to those of diazepam (1.0 mg/kg), a well known anxiolytic drug. On the other hand, the anxiolytic-like effects of obovatol and diazepam were reversed by flumazenil, a benzodiazepine receptor antagonist, suggesting that the anxiolytic-like effects of obovatol were involved in GABA-benzodiazepine receptors complex. Obovatol was muscle relaxant by rota-rod test, but its effect was weaker than diazepam. Spontaneous locomotor activity also was inhibited by obovatol. Obovatol selectively increased the GABA A receptors α 1 subunit expression in amygdala of mouse brain. Obovatol also showed to bind to benzodiazepine receptors competitively in experiments using [3H]flunitrazepam in the cerebral cortex of mouse brain. Moreover, obovatol (10, 20 and 50 μM) increased Cl− influx and the increased Cl− influx was inhibited by flumazenil, in primary cultured neuronal cells and IMR-32 human neuroblastoma cells. These results suggest that obovatol has anxiolytic-like effects, and these pharmacological effects may be mediated by GABA-benzodiazepine receptors-activated Cl− channel opening.