Investigate the involvement of monoaminergic and glutamatergic systems on the antinociceptive and ataxic effects of uliginosin B, which we have already demonstrated to be a promising molecular scaffold to develop new analgesic drugs. Uliginosin B was obtained from hexane extract of aerial parts of Hypericum polyanthemum by chromatographic methods. Uliginosin B antinociceptive and motor coordination effects were evaluated in mice by using hot-plate (15 and 90 mg/kg, i.p.) and rotarod (90 mg/kg, i.p.) tests, respectively. The mechanism of action was investigated through pretreatments with prazosin 1 mg/kg intraperitoneal (α1 receptor antagonist), yohimbine 5 mg/kg intraperitoneal (α2 receptor antagonist), pCPA 300 mg/kg intraperitoneal (serotonin synthesis inhibitor) and MK-801 0.25 mg/kg intraperitoneal (N-methyl-D-aspartic acid receptor antagonist). The antinociceptive effect of uliginosin B (15 and 90 mg/kg, i.p.) was reduced significantly by pCPA and MK-801. Prazosin and yohimbine improved the antinociceptive effect of the highest dose (90 mg/kg, i.p.) of uliginosin B only. The ataxic effect of uliginosin B (90 mg/kg, i.p.) was completely prevented by pretreatment with pCPA or MK-801, but it was unaffected by pretreatment with prazosin or yohimbine. These data confirm the contribution of monoaminergic neurotransmission as well as provide the first evidence of glutamatergic neurotransmission contribution to the uliginosin B effects.