Junctophilin-2 (JP2) is a structural protein that tethers the sarcoplasmic reticulum (SR) to the plasma membrane (PM) within the cardiac dyad, enabling crosstalk between ion channels on the PM and SR, required for excitation-contraction coupling. Contractile regulation of vascular smooth muscle cells (SMCs) also relies on close interactions between the PM and SR, but little is known about the molecular architecture of these peripheral coupling sites (PCS). This study tested the hypothesis that JP2 is critical for maintaining interactions between the SR and PM. C57/Bl6 mice were euthanized according to approved protocols by the IACUC of the University of Nevada, Reno, and the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. We found that mRNA encoding JP2 was present in native, contractile SMCs and JP2 protein expression using the Wes capillary electrophoresis-based immunoassay (N=3). Confocal and super-resolution microscopy showed co-localization of JP2 and type 2 ryanodine receptors (RyR2) in immunolabeled SMCs (n=9 cells, 3 animals). In cerebral artery SMC, RyR2 on the SR are functionally coupled with Ca2+-activated K+ (BK) channels on the PM, a negative feedback system that buffers vasoconstriction. We employed a molecular interference approach using selective morpholinos to knockdown JP2 expression in isolated cerebral pial arteries. JP2 expression was reduced by ~50% compared with the non-targeting control (N=3). 3D live cell deconvolved confocal imaging of the PCS was undertaken using the PM dye (CellMask) and the SR dye (ER-Tracker). SMCs isolated from JP2 targeting morpholinos treated vessels showed a reduction in the volume of PCS compared to SMCs isolated from control vessels (0.73 ± 0.2% vs. 1.84 ± 0.5%, n=9-17 cells, 3 animals/group). Ca2+ release from RyR2 were recorded in pressurised arteries, treated with associated morpholino. Knockdown of JP2 did not affect the frequency, amplitude or kinetics of these Ca2+ events (n=5-6 vessels, 3 animals/group). Electrophysiology was performed to record spontaneous transient outward currents (STOCs), which represents the activation of BK channels by Ca2+ released from RyR2. These studies found that STOCs were absent from SMCs isolated from JP2-targeted morpholinos vessels, whereas STOCs in SMCs from control vessels were unaffected (0.17 ± 0.12 Hz vs. 1.48 ± 0.31 Hz, at -20 mV, n=5-6 cells, 3 animals/group). Finally, the myogenic response was tested between JP2 knockdown and control vessels. JP2 morpholino treated vessels displayed an increased myogenic tone compared to the non-targeting controls (23.19 ± 2.28 % vs. 14.16 ± 2.4 %, at 80 mmHg, n=5-6 vessels, 3 animals/group). This study is the first to show the presence of JP2 in contractile arterial SMCs and it role in maintaining functional coupling between RyR2 and BK channels.