The C4 photosynthetic pathway enriches carbon dioxide in the vicinity of Rubisco, thereby enabling plants to assimilate carbon more efficiently. Three canonical subtypes of C4 exist, named after their main decarboxylating enzymes: NAD-dependent malic enzyme type, NADP-dependent malic enzyme type and phosphoenolpyruvate carboxykinase type. Cleome gynandra is known to perform NAD-ME type C4 photosynthesis. To further assess the mode of C4 in C. gynandra and its manifestation in leaves of different age, total enzyme activities of eight C4-related enzymes and the relative abundance of 31 metabolites were measured. C. spinosa was used as a C3 control. C. gynandra was confirmed as an NAD-ME type C4 plant in mid-aged leaves, whereas a mixed NAD-ME and PEPCK type was observed in older leaves. Young leaves showed a C3-C4 intermediate state with respect to enzyme activities and metabolite abundances. Comparative transcriptome analysis of mid-aged leaves of C. gynandra and C. spinosa showed that the transcript of only one aspartate aminotransferase (AspAT) isoform is highly abundant in C. gynandra. However, the canonical model of the NAD-ME pathway requires two AspATs, a mitochondrial and a cytosolic isoform. Surprisingly, our results indicate the existence of only one highly abundant AspAT isoform. Using GFP-fusion, this isozyme was localised exclusively to mitochondria. We propose a revised model of NAD-ME type C4 photosynthesis in C. gynandra, in which both AspAT catalysed reactions take place in mitochondria and PEPCK catalyses an alternative decarboxylating pathway.