Neural and humoral autonomic mechanisms may be important in the maintenance of blood flow in the masseter muscle (MBF). However, their interactions remain unclear. In this study, we examined interactions between neural and humoral regulation of MBF and investigated the mechanisms mediating these interactions in urethane-anesthetized rats. Stimulation of the adrenal nerve (AN) projecting to the adrenal medulla increased MBF, and this increase was mediated by β-adrenoceptors. Sectioning of the superior cervical sympathetic trunk (CST) significantly inhibited increases in MBF induced by AN stimulation during high activity in the CST, but not during low activity. AN stimulation with clonidine after CST sectioning induced a significant increased in MBF, however phenylephrine had no observable effect. Pretreatment with yohimbine or propranolol significantly inhibited the increase in the MBF. Our results suggest an interaction between β-adrenergic vasodilation evoked by circulating adrenaline and the cervical sympathetic nerves that is mediated by α2-adrenoceptors in the masseter muscle.