Almost all organisms contain the same biosynthetic pathway for the synthesis of l-serine from the glycolytic intermediate, d-3-phosphoglycerate. However, regulation of this pathway varies from organism to organism. Many organisms control the activity of the first enzyme in the pathway, d-3-phosphoglycerate dehydrogenase (PGDH), by feedback inhibition through the interaction of l-serine with the ACT domains within the enzyme. The last enzyme in the pathway, phosphoserine phosphatase (PSP), has also been reported to be inhibited by l-serine. The high degree of sequence homology between Mycobacterium tuberculosis PSP (mtPSP) and Mycobacterium avium PSP (maPSP), which has recently been shown to contain ACT domains, suggested that the mtPSP also contained ACT domains. This raised the question of whether the ACT domains in mtPSP played a functional role similar to that of the ACT domains in PGDH. This investigation reveals that l-serine allosterically inhibits mtPSP by a mechanism of partial competitive inhibition by binding to the ACT domains. Therefore, in mtPSP, l-serine is an allosteric feedback inhibitor that acts by decreasing the affinity of the substrate for the enzyme. mtPGDH is also feedback inhibited by l-serine, but only in the presence of millimolar concentrations of phosphate. Therefore, the inhibition of mtPSP by l-serine would act as a secondary control point for the regulation of the l-serine biosynthetic pathway under physiological conditions where the level of phosphate would be below that needed for l-serine feedback inhibition of mtPGDH.