The Arabidopsis thaliana K+ channel KAT1 has been suggested to have a key role in mediating the aperture of stomata pores on the surface of plant leaves. Although the activity of KAT1 is thought to be regulated by phosphorylation, the endogenous pathway and the primary target site for this modification remained unknown. In the present study, we have demonstrated that the C-terminal region of KAT1 acts as a phosphorylation target for the Arabidopsis calcium-independent ABA (abscisic acid)-activated protein kinase SnRK2.6 (Snf1-related protein kinase 2.6). This was confirmed by LC-MS/MS (liquid chromatography tandem MS) analysis, which showed that Thr306 and Thr308 of KAT1 were modified by phosphorylation. The role of these specific residues was examined by single point mutations and measurement of KAT1 channel activities in Xenopus oocyte and yeast systems. Modification of Thr308 had minimal effect on KAT1 activity. On the other hand, modification of Thr306 reduced the K+ transport uptake activity of KAT1 in both systems, indicating that Thr306 is responsible for the functional regulation of KAT1. These results suggest that negative regulation of KAT1 activity, required for stomatal closure, probably occurs by phosphorylation of KAT1 Thr306 by the stress-activated endogenous SnRK2.6 protein kinase.