Rutin is a bioflavonoid found in medicinal plants used to reduce anxiety. Evidence is lacking of rutin’s anxiolytic-like activity, putative mechanism(s) of action, and neural sites of effects. The basolateral amygdala (BLA) is the main brain region that regulates anxiety, through GABAA/benzodiazepine (BDZ) receptors, which are modulated by flavonoids. Therefore, the main aim of this study was to investigate whether the anxiolytic-like effect of rutin involves GABAA/BDZ receptors in the BLA. Rutin was administered systemically (30–1000 mg/kg, intraperitoneal) or microinjected into the BLA (16 nmol/4 µl, intracerebral), and its effects were assessed in the elevated plus-maze and open-field tests. Diazepam (1 mg/kg, intraperitoneal, or 7 nmol/4 µl, respectively) was used as a positive control. The mechanism of action was studied using flumazenil (BDZ antagonist, 5 mg/kg, intraperitoneal, or 7 nmol/4 µl, intracerebral) or picrotoxin (chloride channel GABAA antagonist, 0.3 nmol/4 µl, intracerebral). Rutin, administered systemically or intra-amygdala, induced anxiolytic-like responses, similar to those of diazepam. The effect of diazepam was completely blocked by flumazenil, which also partly antagonized the effects of systemic rutin. By contrast, flumazenil exerted no effect and picrotoxin had only a partial action when rutin was infused in the BLA. These results suggest that the anxiolytic-like effect of rutin in the BLA involves GABAergic neurotransmission that is not associated with BDZ receptors.