Reduced expression of glutamate decarboxylase 67 (GAD67), encoded by Gad1 gene, is a consistent finding in postmortem brains of patients with schizophrenia, bipolar disorder and major depressive disorder. However, the neurobiological consequences of GAD67 dysfunction in mature brains are not fully understood because the homozygous Gad1 knockout is lethal in newborn mice. Here, we developed Gad1^{tTA/STOP-tetO} biallelic knock-in mice using Gad1^STOP-tetO and Gad1^tTA knock-in mice, and compared them with Gad1^+/+ mice. The expression level of GAD67 protein in brains of Gad1^{tTA/STOP-tetO} mice treated with doxycycline (Dox) was decreased by approximately 90%. The GABA content was also decreased in the brains of Dox-treated Gad1^{tTA/STOP-tetO} mice. In the open-field test, Dox-treated Gad1^{tTA/STOP-tetO} mice exhibited hyper-locomotor activity and decreased duration spent in the center region. In addition, acoustic startle responses were impaired in Dox-treated Gad1^{tTA/STOP-tetO} mice. These results suggest that global reduction in GAD67 elicits behavioral impairments in mice. These GAD67 knockdown mice will be useful for elucidating the neurobiological mechanisms of emotional abnormalities, such as anxiety symptoms associated with psychiatric disorders.