Cortical spreading depression (CSD) is an important disease model for migraine and stroke. Previous studies showed that before the pial artery large dilation which were widely observed in CSD, there is also a small constriction occurring in pial arteres. However, the mechanisms contributing to the regulation of this cerebrovascular response remain unknown. Optical intrinsic signal imaging(OISI) at 550 nm wavelength was used to monitor the responses of the pial arteries during pinprick induced CSD following the application of a K_ATP antagonist glibenclamide (glyb). By applying the glyb, the initial slight constriction (ISC) is obviously reduced, most of the ISC (10 μmol/L: 74.5%; 100 μmol/L: 96.2%) even completely restrained. And the peak of large dilation (LD) in response to the pial arteries was enhanced to (53.8±19.3) % and (59.8±19.6) % of the control with 10 μmol/L and 100 μmol/L glyb, respectively. It can be suggested that K_ATP in nerve cells of pre and post-synaptically plays a lead role in inhibiting CSD-associated hyperemia.