Objective: We investigated the role of intracellular steady state pH in smooth muscle and endothelial cells for vascular function and blood pressure regulation. Methods: Intracellular pH regulation was impeded by knockout of the electroneutral Na⁺,HCO₃^--cotransporter NBCn1 (slc4a7). The function of isolated arteries was assessed using myography combined with fluorescence microscopy and NO-sensitive electrodes. Rho-kinase activity was investigated by an in vitro assay. Blood pressure was measured with a tail-cuff approach. Results: Knockout of NBCn1 caused a reduced intracellular pH in mesenteric artery smooth muscle and endothelial cells where it abolished all Na⁺,HCO₃^--cotransport. Isolated arteries from NBCn1 knockout mice had reduced acetylcholine induced, NO-mediated relaxations and rho-kinase dependent smooth muscle Ca²⁺-sensitivity. Arterial blood pressure was 9 mmHg higher in NBCn1 knockout compared to wild type mice. The NBCn1 knockout mice displayed an attenuated blood pressure increase to NO-synthase inhibition, a reduced blood pressure decrease to rho-kinase inhibition and were resistant to developing hypertension during angiotensin II infusion. The increase of extracellular NO in the presence of acetylcholine was reduced in arteries from NBCn1 knockout mice while dilation to the NO-donor S-nitroso-N-acetylpenicillamine, the acetylcholine-induced endothelial Ca²⁺-increase and the endothelial NO-synthase expression were unaffected. Rho-kinase activity was reduced at low pH. In the absence of CO₂/HCO₃^-, no differences were observed between isolated arteries from NBCn1 knockout and wild type mice. Conclusions: Using a knockout mouse to induce sustained intracellular acidification, we show a novel interaction between smooth muscle cell and endothelial cell steady state intracellular pH, vascular function and blood pressure regulation.