Microglia, the immune cells of the central nervous system (CNS), are busy and vigilant housekeepers in the adult brain. The main candidate as a chemoattractant for microglia at damaged site is adenosine triphosphate (ATP). Some neuropeptides, such as bradykinin and galanin, are also chemoattractants for microglia with different mechanism from that of ATP. In the present study, we found that ATP-induced microglial migration and morphological changes were regulated by orexin. Migration of cultured mice microglia was monitored with time lapse video microscopy system and Boyden chamber. Morphological change and the number of microglia in vivo were analyzed immunocyto- or immunohisto-chemically. Receptor expression was analyzed by RT-PCR and western blotting. Among various neuropeptides, orexin did not affect microglial migration by itself but inhibited ATP-induced microglial migration. Mouse microglia expresses mainly orexin receptor 1 (OXR1). From pharmacological studies and using orexin receptor (OXR1 and OXR2) knock-out (KO) mice, it was observed that orexin-A activated Gs-coupling OXR1 in microglia, competing with Gi-coupling P2Y receptors. Orexin increased intracellular cyclic adenosine monophosphate (cAMP) in cultured microglia and indeed ATP-induced migration was inhibited by membrane permeable cAMP. On the other hand, orexin-A did not affect ATP-activated PI3K/Akt signaling. From morphological analyses, orexin A also inhibited ATP-induced microglial ruffling. Using in vivo lesion models, microglial accumulation around the lesion was inhibited in OXR2-KO mice, where OXR1 was somehow up-regulated. Microglial motility and morphological change induced by ATP, one of the neurotransmitters, was inhibited by orexin. These results may help understanding unknown function of neuropeptides in microglia and their physiological and pathophysiological roles.