In guard cells, activation of anion channels (Ianion) is an early event leading to stomatal closure. Activation of Ianion has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). However, the dynamics of the action of [Ca2+]i on Ianion has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca2+]i dynamics of Ianion in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca2+]i using Fura-2 fluorescence imaging. We found that Ianion rises with [Ca2+]i only at concentrations substantially above the mean resting value of 125 ± 13 nm, yielding an apparent Kd of 720 ± 65 nm and a Hill coefficient consistent with the binding of three to four Ca2+ ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of Ianion activity, but without a dependence of the current on [Ca2+]i. The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca2+]i sensitivity of Ianion, displacing the apparent Kd for [Ca2+]i to 573 ± 38 nm. These findings support previous evidence for different modes of regulation for Ianion, only one of which depends on [Ca2+]i, and they underscore an independence of [Ca2+]i from protein (de-)phosphorylation in controlling Ianion. Most importantly, our results demonstrate a significant displacement of Ianion sensitivity to higher [Ca2+]i compared with that of the guard cell K+ channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.