In plant cells, anion channels and transporters are essential for key functions such as nutrition, resistance to biotic or abiotic stresses, and ion homeostasis. In Arabidopsis, members of the chloride channel (CLC) family located in intracellular organelles have been shown to be required for nitrate homeostasis or pH adjustment, and previous results indicated that AtCLCc is involved in nitrate accumulation. We investigated new physiological functions of this CLC member in Arabidopsis. Here we report that AtCLCc is strongly expressed in guard cells and pollen and more weakly in roots. Use of an AtCLCc:GFP fusion revealed localization to the tonoplast. Disruption of the AtCLCc gene by a T-DNA insertion in four independent lines affected physiological responses that are directly related to the movement of chloride across the tonoplast membrane. Opening of clcc stomata was reduced in response to light, and ABA treatment failed to induce their closure, whereas application of KNO3 but not KCl restored stomatal opening. clcc mutant plants were hypersensitive to NaCl treatment when grown on soil, and to NaCl and KCl in vitro, confirming the chloride dependence of the phenotype. These phenotypes were associated with modifications of chloride content in both guard cells and roots. These data demonstrate that AtCLCc is essential for stomatal movement and salt tolerance by regulating chloride homeostasis.