Glyceraldehyde-3-phosphate dehydrogenases catalyze key steps in energy and reducing power partitioning in cells of higher plants. Phosphorylated non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) present in heterotrophic cells of wheat (Triticum aestivum) was activated up to 3-fold by MgCl2. The effect was not observed with the non-phosphorylated enzyme found in leaves. The divalent cation also affected the response of the enzyme from endosperm and shoots to adenine nucleotides and inorganic pyrophosphate. Gel filtration chromatography, co-immunoprecipitation followed by immunostaining, and the use of a phosphopeptide containing a canonical binding motif showed that MgCl2 actually disrupted the interaction between GAPN and a 14-3-3 regulatory protein. After interaction with 14-3-3, phosphorylated GAPN exhibits a 3-fold lower Vmax and higher sensitivity to inhibition by ATP and pyrophosphate. Results suggest that GAPN is a target for regulation by phosphorylation, levels of divalent cations, and 14-3-3 proteins. The regulatory mechanism could be critical to maintain levels of energy and reductants in the cytoplasm of heterotrophic plant cells.