Although Kir6.2, one of the subunits composed ATP-sensitive potassium (K_ATP) channels, are widely distributed in the brain, the roles in emotional behavior are not yet fully understood. Here we investigated the behavioral characteristics of Kir6.2-deficient (Kir6.2^-/-) mice. Kir6.2^-/- mice showed anxiety-like behavior in the elevated-plus maze test and light-dark test, especially, it was prominent in females. Immunohistochemical studies showed that Kir6.2 was co-localized with tryptophan hydroxylase (TPH) in the dorsal raphe nuclei and tyrosine hydroxylase (TH) in the ventral tegmental area/locus coeruleus. Interestingly, TPH expression in the midbrain was significantly elevated in female Kir6.2^-/- mice. These results suggest that Kir6.2 expressed in serotonergic neurons could play a key role in emotional behavior. Furthermore, we investigated whether pharmacological blockade of K_ATP channels affects the emotional behavior. Mice that had been injected intracerebroventricularly with glibenclamide, a selective K_ATP channel blocker, showed anxiety-like behavior in the elevated-plus maze test. These results confirm a critical role of K_ATP channels in regulation of emotional behavior.