Mycobacterium tuberculosis (Mtb) is the leading bacterial killer in the world, taking the lives of over one million people every year for centuries. Mtb has evolved distinct metabolic adaptations to enable it to persist in our immune system instead of being eradicated by it. Part of this adaptation involves the modulation of flux in the tricarboxylic acid (TCA) cycle-glyoxylate shunt junction according to its nutrient availability. This junction has been highlighted as a critical metabolic node in Mtb, and studies have shown a fine balance in the flux between the two pathways. A perturbation of this balance impacts the growth and survival of Mtb, but it is not known how such balance is achieved. In this study, the regulatory mechanisms behind the balance in this junction were investigated by analysing the regulation of the gatekeeping enzymes into each pathway – isocitrate lyase (ICL) for the glyoxylate shunt and isocitrate dehydrogenase (ICD) for the TCA cycle. An NMR-based in vitro model was created to observe the flux between the two pathways. This is the first instance of an experimental illustration of the flux between competing pathways in a metabolic junction. On the gene level, ICL was significantly upregulated when placed in pathogenically relevant conditions, while ICD levels did not fluctuate significantly. On the protein level, metabolites from upstream pathways were screened for their ability to affect the activity of the two enzymes. This resulted in novel metabolite activators of ICL and ICD. The effect of the ICD activators was also found to be dependent on ICL activity. Protein NMR and enzyme kinetic analyses illustrated the mechanism of activation behind these metabolites. Metabolomics studies in Mtb cells highlighted the importance of achieving a stable level of these metabolites in-cell. Overall, these results can be used to design a multi-pronged approach to target the junction from different levels for a more effective eradication of Mtb.