Objective: We studied the effect of NHE1 knockout on intracellular pH (pHi) regulation in vascular smooth muscle (VSMCs) and endothelial cells (ECs), its impact on mesenteric artery structure and function and its role for blood pressure regulation. Methods: Vessel myography was combined with fluorescence microscopy. Histology was used for stereological analyses. Blood pressure was measured by a tail-cuff approach. Results: Na/H-exchange activity was abolished in VSMCs and ECs from 4-5 weeks old NHE1 knockout mice. In VSMCs and ECs, steady-state pHi was reduced (0.3 and 0.6 pH-units, respectively) by NHE1 knockout in the absence of CO2/bicarbonate but unaffected in the presence of CO2/bicarbonate. Active tension development to norepinephrine (NE) and K-depolarizations were approximately 30% smaller in arteries from NHE1 knockout compared to wild type mice and the media thickness proportionally reduced. Media cross-sectional area was diminished in arteries from NHE1 knockout mice owing to a reduced VSMC cross-sectional area and volume. Active force production per VSMC was reduced whereas active force production per VSMC cross-sectional area and the VSMC Ca²⁺-response to NE were unaffected in arteries from NHE1 knockout mice. Mean arterial blood pressure was reduced from 109±8 mmHg in wild type to 84±4 mmHg in NHE1 knockout mice while heart rate was unaffected. Omission of CO2/bicarbonate inhibited NO-synthase dependent relaxations to acetylcholine in arteries from NHE1 knockout but not wild type mice. The EC Ca²⁺-response to acetylcholine and vasorelaxation to the NO-donor SNAP were unaffected by removal of CO2/bicarbonate. Conclusion: We conclude that NHE1 is the only Na/H-exchanger in ECs and VSMCs of mouse mesenteric arteries. NHE1 knockout causes hypotrophy of VSMCs and reduces active arterial tension development and blood pressure. Steady-state pHi is reduced but only in the absence of CO2/bicarbonate. We suggest that endothelial acidification inhibits endothelial NO production.