Background: Cystic fibrosis transmembrane conductance regulator (CFTR) has been associated with vascular tone and blood pressure (BP), however, its role in the genesis of hypertension remains elusive. In the present study, we investigated the regulating effect of CFTR on angiotensin II (Ang II) -induced hypertension and defined the molecular role of CFTR in vasoconstriction. Results: We found that CFTR mRNA and protein expression were markedly down-regulated in the arteries from Ang II induced hypertensive animals. During the development of hypertension, BP of Cftr{}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}} mice was significantly higher than that of Cftr{}^{\mathrm{+}\mathit{}\mathrm{/}\mathrm{+}} mice. Arteries from Cftr{}^{\mathrm{-}\mathit{}\mathrm{/}\mathrm{-}} mice or pre-incubated with CFTR specific inhibitor CFTR(inh)-172 exhibited a greater contractile response to Ang II. In vascular smooth muscle cells (VSMCs), the phosphorylation of myosin light chain (MLC), which is the core of VSMCs contraction, was negatively modulated by CFTR. Furthermore, intracellular Ca{}^{2+} concentration ([Ca{}^{2+}]{}_i) rise in response to Ang II was negatively modulated by CFTR, while no alteration was observed in resting VSMCs. Ras homolog family member A/Rho-associated protein kinase (RhoA/Rock) mediated phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a regulator of MLC phosphorylation, was negatively modulated by CFTR in both resting and Ang II-stimulated VSMCs. Conclusions: This study demonstrates that CFTR is a negative regulator of vasoconstriction and hypertension, and the underlying mechanism contains two possible pathways: (1) in resting VSMCs, CFTR altered MLC phosphorylation through RhoA/Rock pathway; (2) in Ang II stimulated VSMCs, the regulating effect was mediated by both Ca{}^{2+} influx and RhoA/Rock mediated pathway.