Angiotensin II (Ang II) has a large impact in cardiovascular physiology and serves as a potent pharmacological target in the treatment of hypertension. In coronary arteries, Ang II has been reported to inhibit the activity of the large conductance calcium-activated potassium (BK) channel, a primary determinant of vascular tone. The present study aims to investigate: 1) whether Ang II regulates BK channels in the renal artery, as the kidney plays a pivotal role in maintaining normal systemic blood pressure; and 2) the underlying molecular mechanisms of the regulation. Male three month old Sprague Dawley rats were used. Animal protocols received institutional approval. We found in freshly isolated rat renal arterial smooth muscle cells (SMCs) that 1 μM Ang II reduced the iberiotoxin-sensitive whole-cell BK currents by 44 ± 8.7%. This inhibitory effect was fully prevented by 10 µM losartan, an antagonist of Angiotensin II type 1 receptor (AT1R) but remained intact in the presence of 500 µM GDPβS, an inhibitor of G-protein activation. Results from whole-cell patch clamp in HEK293T cells further demonstrated that the presence of AT1R is required for the inhibitory effect of Ang II on BK channel activity. Moreover, AT1R was found in physical association with BK channel α-subunit (BKα, Slo1) by co-immunoprecipitation analysis. Consistent with the biochemical results, we observed a substantial colocalization of AT1R and BKα at the plasma membrane using live labeling and confocal microscopy. In keeping with a tight coupling between AT1R and BK channels resulting in an overall inhibitory effect on BK channels, electrophysiological studies revealed that the sole expression of AT1R (in the absence of Ang II) shifted the half activation potential of BK channels as much as ~32 mV towards more positive membrane potentials. Finally, Ang II induced internalization of AT1R together with a dramatic increase of the cytoplasmic presentation of BK channels in HEK293T cells. These results strongly suggest that AT1R is the mediator of the Ang II effect on BK channels from coronary and renal arterial SMCs, and unravel a protein-protein interaction-based mechanism independent of G-protein activation by which AT1R modifies BK channel properties and microlocalization.