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C4 photosynthesis is a complex trait that involves the efficient movement of carbon between mesophyll and bundle sheath cells, but gaps remain in our understanding on how protein phosphorylation regulates the relative flux through malate or aspartate in NADP-malic enzyme subtypes. Using mass spectrometry, 20 phosphosites in NADPmalic enzyme (ME), 9 phosphosites in aspartate aminotransferase (AAT) and 12 phosphosites in alanine aminotransferase were identified in the C4 plant model Setaria viridis. In silico analysis shows that NADP-ME phosphorylation at Ser-343 may regulate NADP+ binding. NADP-ME from illuminated leaves of Zea mays exhibited a higher affinity for L-malate at pH 8.4. High in vitro activity at pH 7.4 in illuminated leaves of Sorghum bicolor, and no change in affinity for L-malate at pH 7.4 or pH 8.4 reveals dependence on enzyme activity immediately after the onset of light. It is speculated that the same protein may be differently regulated in each grass species. The activation of AAT by C4-acids also differed among the NADP-ME subtypes. Light activation in the presence of L-alanine and L-malate was only evident in Zea mays, whereas L-malate and L-alanine inhibited the activity in Sorghum bicolor and L-malate inhibited activity in Setaria viridis. In vitro activity assays also demonstrated that the affinity for L-aspartate was higher in darkened conditions and this activity may not be associated with C4 photosynthesis. The work presented in this study shows that there are distinct interspecies differences in the properties of NADP-ME and AAT. These observations are discussed in terms of a regulatory mechanism that controls the relative flux through malate and aspartate in a dual-decarboxylation system of Zea mays, but not in Sorghum bicolor. Together, these findings add to our understanding of the regulation of C4 photosynthesis, thereby advancing the wider objective of engineering the C4 trait into existing C3 crops.