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Present study was carried out to investigate the ‘anxiolytic-like’ effect of pyridoxine in mice. Pyridoxine (90, 180 and 360 mg/kg) was administered by intraperitoneal (i.p.) route to the experimental mice and anxiety-related behavior was evaluated by light and dark box (LDB) and elevated plus maze (EPM) models. Glutamate, GABA and nitrite levels were also determined in the isolated whole brain of mice. It was observed that pyridoxine (180 mg/kg, i.p.) exerted ‘anxiolytic-like’ effect in mice in EPM and LDB models. Also, there was a significant increase in the levels of GABA whereas; the levels of glutamate and nitrite were decreased as compared to the control group. Administration of pentamethylene tetrazole (PTZ; 20 mg/kg, i.p.) exerted anxiogenic effects in mice, but the combination of PTZ and pyridoxine (180 mg/kg, i.p.) abolished the ‘anxiolytic-like’ effect of pyridoxine, thereby, suggesting the possible role of GABA in the ‘anxiolytic-like’ effect of pyridoxine in mice. Further, the influence of NO-sGC-cGMP pathway was investigated by administering the sub-effective dose of pyridoxine in combination with sub-threshold doses of NO modulators i.e. l-arginine (50 mg/kg, i.p.; NO donor); methylene blue (1 mg/kg, i.p.; NO and soluble guanylate cyclase inhibitor) and sildenafil (1 mg/kg, i.p.; phosphodiesterase inhibitor and cGMP modulator). It was observed that the ‘anxiolytic-like’ effect of pyridoxine in mice was counteracted by the NO donor and potentiated by the NO inhibitors. Thus, the present study confirmed the involvement of GABAergic and NO-sGC-cGMP pathway in the ‘anxiolytic-like’ effect of pyridoxine in mice.Pyridoxine exerted anxiolytic like effects in mice in EPM and LDB.Pyridoxine administration increased the levels of GABA and reduced the levels of glutamate and nitrite in the brain of mice.PTZ treatment in combination with pyridoxine abolished the anxiolytic like effects of pyridoxine in mice.NO-sGC-cGMP pathway influenced the anxiolytic like effects of pyridoxine in mice.