We demonstrated that pannexin1 (Panx1) channels are expressed in the smooth muscle cells of small arteries where they participate in α1D adrenergic receptor (α1DAR)-mediated vasoconstriction1. In this paper, we also showed that ATP is released from vascular smooth muscle cells via Panx1 channels upon phenylephrine (PE) stimulation. Since the α1DAR-mediated constriction as well as ATP signaling are key in the regulation of peripheral resistance and blood pressure, we hypothesized that Panx1 could participate in blood pressure regulation. Therefore, we created an inducible conditional KO mouse model where Panx1 is deleted specifically in smooth muscle cells by breeding smooth muscle myosin heavy chain (SMMHC) – CreERT2+ mice with Panx1fl/fl mice. After 10 days of tamoxifen injections, the expression of Panx1 in arterial smooth muscle cells was abolished and the vasoconstriction of small arteries in response to PE was significantly reduced. Using radiotelemetry, we measured a reduction in the mean arterial pressure in these mice after tamoxifen injections. Because Panx1 is activated by the α1DAR signaling cascade and because the channel and the receptor are closely located at the plasma membrane of arterial smooth muscle cells, we hypothesized that disruption of the Panx1/α1DAR signaling pathway would alter vasoconstriction. In order to specifically inhibit Panx1 activation by the α1DAR, we designed a peptide mimicking the intracellular loop (IL) region of Panx1, which is enriched in proline, making this region more susceptible to interact with protein partners for intracellular signaling. When treated with the IL peptide, pressurized small arteries exhibited decreased vasoconstriction to PE. In parallel, we performed electrophysiology on HEK cells co-transfected with Panx1 and α1DAR plasmids and confirmed PE-induced Panx1 activation in vitro by measuring an increase of Panx1 current and ATP release upon PE stimulation. The PE-induced Panx1 activation was also altered by Panx1 IL peptide. To further investigate the key amino acids of Panx1 intracellular loop in PE-induced Panx1 activation, we created specific mutants of Panx1 in the region mimicked by the IL peptide and cotransfected these mutants in HEK cells along with the α1DAR plasmid. Both ATP release and Panx1 currents were decreased when the amino acids YLK in position 198 to 200 were mutated to alanine, showing their essential role in PE-induced Panx1 activation. In summary, our results show that Panx1 plays an essential role in the α1DAR-mediated constriction by releasing ATP and thus in the regulation of blood pressure. Furthermore, we have identified a key region in Panx1 intracellular loop that is essential in the PE-induced activation of Panx1. This work provides the basis for a novel understanding of blood pressure control by the sympathetic nervous system.