The canonical Gα subunit of the heterotrimeric G protein complex from wheat (Triticum aestivum), GA3, and the calcium-binding protein, Clo3, were revealed to interact both in vivo and in vitro and Clo3 was shown to enhance the GTPase activity of GA3. Clo3 is a member of the caleosin gene family in wheat with a single EF-hand domain and is induced during cold acclimation. Bimolecular Fluorescent Complementation (BiFC) was used to localize the interaction between Clo3 and GA3 to the plasma membrane (PM). Even though heterotrimeric G-protein signaling and Ca²⁺ signaling have both been shown to play a role in the response to environmental stresses in plants, little is known about the interaction between calcium-binding proteins and Gα. The GAP activity of Clo3 towards GA3 suggests it may play a role in the inactivation of GA3 as part of the stress response in plants. GA3 was also shown to interact with the phosphoinositide-specific phospholipase C, PI-PLC1, not only in the PM but also in the endoplasmic reticulum (ER). Surprisingly, Clo3 was also shown to interact with PI-PLC1 in the PM and ER. In vitro analysis of the protein-protein interaction showed that the interaction of Clo3 with GA3 and PI-PLC1 is enhanced by high Ca²⁺ levels. Three-way affinity characterizations with GA3, Clo3 and PI-PLC1 showed the interaction with Clo3 to be competitive, which suggests that Clo3 may play a role in the Ca²⁺-triggered feedback regulation of both GA3 and PI-PLC1. This hypothesis was further supported by the demonstration that Clo3 has GAP activity with GA3.