The present study investigated the anxiolytic-like effects of spinosin, one of the major flavonoids in Ziziphi Spinosae Semen (ZSS), in experimental models of anxiety compared with a known anxiolytic, diazepam. Repeated treatment with spinosin (2.5 and 5 mg/kg/day, p.o.) significantly increased the percentage of entries into and time spent on the open arms of the elevated plus maze compared with the control group. In the light/dark box test, spinosin exerted an anxiolytic-like effect at 5 mg/kg. In the open-field test, 5 mg/kg spinosin increased the number of central entries. Spinosin did not affect spontaneous activity. The anxiolytic-like effects of spinosin in the elevated plus maze, light/dark box test, and open field test were blocked by the γ-aminobutyric acid-A (GABAA) receptor antagonist flumazenil (3 mg/kg, i.p.) and 5-hydroxytryptamine-1A (5-HT1A) receptor antagonist WAY-100635 (1 mg/kg, i.p.). These results suggest that spinosin exerts anxiolytic-like effects, and its mechanism of action appears to be modulated by GABAA and 5-HT1A receptors.