Abstract This study investigated the anxiolytic-like effects of 4-O-methylhonokiol, a neolignan compound of Magnolia officinalis, by using the experimental paradigms of anxiety and compared the results with those of a known anxiolytic, diazepam. A single treatment with 4-O-methylhonokiol (0.1, 0.2, and 0.5 mg/kg, p.o.) or treatment for 7 days (0.5 mg/kg in drinking water) increased the percentage of time spent in the open arms and the number of open arms entries in the elevated plus-maze test. However, the 4-O-methylhonokiol-increased percentage of time spent in the open arm was abolished by treatment with flumazenil, a benzodiazepine receptor antagonist (10 mg/kg). 4-O-Methylhonokiol also increased the number of head dips in the hole-board test, but decreased locomotor activity. Molecular experiments revealed that the α1-subunit of γ-aminobutyric acid (GABA) type A receptors was overexpressed in the cortex of brains of mice after treatment with 4-O-methylhonokiol for 7 days. In addition, 4-O-methylhonokiol also increased chloride influx in cultured cortical cells. It is concluded that 4-O-methylhonokiol may have anxiolytic-like effects and that these effects may be mediated by GABAergic transmission with the increase of Cl(-) channel opening.