The aim of the present study was to investigate the role of GABAergic and nitriergic modulation in the antianxiety effect of thymoquinone, a major constituent of nigella sativa, in mice under unstressed and stressed conditions. Thymoquinone (10 and 20 mg/kg), methylene blue (1 mg/kg) and diazepam (2 mg/kg) were administered followed by behavioral testing using an elevated plus maze, the light/dark test and the social interaction test in both unstressed and stressed mice (mice subjected to 6 h immobilization). The effects of the above-mentioned drugs on plasma nitrite, a stable metabolite of nitric oxide (NO) and brain GABA content were also studied. Diazepam (2 mg/kg) produced significant anxiolytic-like effects only in unstressed mice. However, diazepam significantly increased the GABA content in both unstressed and stressed mice as compared with their respective control groups. Thymoquinone (10 and 20 mg/kg) produced significant antianxiety effects in unstressed mice without altering nitrite levels, but only the higher dose (20 mg/kg) of thymoquinone increased the GABA content in unstressed mice. In stressed mice, thymoquinone (20 mg/kg) showed anxiolytic effects, with a significant decrease in plasma nitrite and reversal of the decreased brain GABA content. Pre-treatment with methylene blue enhanced the antianxiety effect of thymoquinone in both unstressed and stressed mice. Therefore, the present study suggests an involvement of NO-cGMP and GABAergic pathways in the anxiolytic-like activity of thymoquinone.