Methamphetamine has a central nervous system stimulant action and causes hyperactivity and elevation of body temperature. Recently, it has been shown that orexin system in the brain plays a key role of stress-related autonomic physiological response such as tachycardia and hyperthermia. In a orexin neuron, various neurotransmitters are co-localized with orexin. Among them, glutamate is focused on since its importance in autonomic physiological response to stress has been suggested. The aim of this study is to explore the role of glutamate as a co-transmitter from orexin neurons in methamphetamine-induced autonomic physiological response. We used a genetically-modified mouse (vGLUT2-KO) in which vGLUT2 is deficient only in orexin neurons and thus glutamate cannot be released from the orexin neurons and a wild type mouse (WT) as a control group. We implanted a telemetry probes to measure heart rate, body temperature and locomotor activity under isoflurance anesthesia (2%, inhalation). After at least one-week recovery period, we examined the effect of methamphetamine (0.5, 2, 5mg/kg, i.p.) on these parameters in conscious free-moving condition. In both groups of vGLUT2-KO and WT mice, methamphetamine caused an increase in body temperature as well as locomotor activity dose-dependently (P<0.05, n=7, Pearson). The amplitude of increases in body temperature as well as activity was significantly greater in vGLUT-KO than in WT mice (by 2.2±0.7 vs by 0.5±0.5 degree Celsius, 0.5mg/kg, n=7, P<0.05, Bonferroni). These results suggest a possible suppressive action of glutamate co-localized in orexin neurons on hyperthermia and hyperactivity induced by methamphetamine.