Big-conductance, calcium-activated potassium (BK) channels are important for numerous physiological responses, including relaxation of vascular smooth muscle cells (SMCs). The activity of BK channels can be regulated by several signaling molecules. Here we provide biochemical evidence showing that FK506 binding protein 12.6 (FKBP12.6), an endogenous molecule known to regulate ryanodine receptors/calcium release channels, is physically associated with the BK channel α subunits in mouse cerebral arteries. Inside-out single channels recordings show that application of FK506 to remove FKBP12.6 significantly decreases the open probability of BK channels in freshly isolated mouse cerebral artery SMCs. The effect of FK506 is concentration-dependent. Similar to chemical removal of FKBP12.6 with FK506 exposure, genetic removal of FKBP12.6 with gene deletion produces an inhibitory effect on the activity of single BK channels as well. FKBP12.6 gene deletion also reduces the sensitivity of BK channels to voltage and calcium. Consistent with these results, agonist-evoked vasoconstriction is augmented in isolated arteries from FKBP2.6 gene deletion mice. Moreover, blood pressure is higher in FKBP12.6 gene deletion mice than control mice. In conclusion, our findings for the first time demonstrate that FKBP12.6 is associated with BK channels and regulates the channel functions, which may play an important role in controlling vascular tone and blood pressure.