Arteries and arterioles exhibit myogenic tone, a partially constricted state that allows further constriction or dilation in response to moment-to-moment fluctuations in blood pressure. The vascular endothelium that lines the internal surface of all blood vessels controls a wide variety of essential functions, including the contractility of the adjacent smooth muscle cells by providing a tonic vasodilatory influence. Studies conducted on large (pial) arteries on the surface of the brain have shown that estrogen lowers myogenic tone in female mice by enhancing nitric oxide (NO) release from the endothelium, however, whether this difference extends to the intracerebral microcirculation remains ambiguous. The existing incomplete picture of sex differences in cerebrovascular physiology combined with a deficiency in treatments that fully restore cognitive function after cerebrovascular accidents places heavy emphasis on the necessity to investigate myogenic tone regulation in the microcirculation from both male and female mice. We hypothesized that sex-linked hormone regulation of myogenic tone extends its influence on the microcirculation level, and sought to characterize it in isolated arterioles from the hippocampus, a major cognitive brain area. Using diameter measurements both in vivo (acute cranial window vascular diameter) and ex vivo (pressure myography experiments), we measured lower myogenic tone responses in hippocampal arterioles from female than male mice. By using a combined surgical and pharmacological approach, we found myogenic tone in ovariectomized (OVX) female mice matches that of males, as well as in endothelium-denuded arterioles. Interestingly, eNOS inhibition induced a larger constriction in female arterioles but only partially abolished the difference in tone. We identified that the remnant difference was mediated by a higher activity and expression of the small-conductance Ca 2+ -sensitive K + (SK) channels. Collectively, these data indicate that eNOS and SK channels exert greater vasodilatory influence over myogenic tone in female mice at physiological pressures.