Leaf stomata close in response to high carbon dioxide levels and open at low CO₂. CO₂ concentrations in leaves are altered by daily dark/light cycles, as well as the continuing rise in atmospheric CO₂. Relative to abscisic acid and blue light signaling, little is known about the molecular, cellular, and genetic mechanisms of CO₂ signaling in guard cells. Interestingly, we report that repetitive Ca²⁺ transients were observed during the stomatal opening stimulus, low [CO₂]. Furthermore, low/high [CO₂] transitions modulated the cytosolic Ca²⁺ transient pattern in Arabidopsis guard cells (Landsberg erecta). Inhibition of cytosolic Ca²⁺ transients, achieved by loading guard cells with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid and not adding external Ca²⁺, attenuated both high CO₂-induced stomatal closing and low CO₂-induced stomatal opening, and also revealed a Ca²⁺-independent phase of the CO₂ response. Furthermore, the mutant, growth controlled by abscisic acid (gca2) shows impairment in [CO₂] modulation of the cytosolic Ca²⁺ transient rate and strong impairment in high CO₂-induced stomatal closing. Our findings provide insights into guard cell CO₂ signaling mechanisms, reveal Ca²⁺-independent events, and demonstrate that calcium elevations can participate in opposed signaling events during stomatal opening and closing. A model is proposed in which CO₂ concentrations prime Ca²⁺ sensors, which could mediate specificity in Ca²⁺ signaling.