Abstract: Alteration of neuropeptide Y (NPY) in serum may cause sleep disturbance, circadian rhythm disorder, and/or even emotional illness. γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter and has been widely used for reduction of depression, anxiety, and/or psychological stress since it can raise the total or parasympathetic nerve activities in the mammalian central nervous system. However, drawbacks including short half-life in vivo and low absorption efficiency make synthetic GABA less usability in drug formulation, while it is more effective to uptake GABA through long term/regular daily diet. In this study, we successfully produced GABA-enriched rice by inoculating grain seeds of Taiwanese rice with Bacillus amyloliquefaciens (B. amyloliquefaciens) during germination, and demonstrated that the B. amyloliquefaciens-inoculated rice (BAR) can grow with a normal root/sprout length but significantly increased germination ratio compared to the rice without B. amyloliquefaciens treatment. Based on the animal study in association with the hole board test, our data showed that the level of serum NPY as well as the number of head-dips of the mice treated with the BAR for 8 days significantly enhanced 2 folds (P < 0.01) compared to the mice without GABA, but such BAR-induced efficacies dramatically vanished as their gastrointestinal GABAB receptor and/or vagus nerves were removed through use of GABA receptor antagonist or vagotomy, respectively. These results manifested the potential of the BAR on anxiolytic applications and unveiled likely mediators for GABA-induced anxiolytic activity.