Biosynthesis and acquisition of nutrients during infection are integral to pathogenesis. Members of a metabolic pathway, the glycine cleavage system, have been identified in virulence screens of the intracellular bacterium Francisella tularensis but their role in pathogenesis remains unknown. This system generates 5,10-methylenetetrahydrofolate, a precursor of amino acid and DNA synthesis, from glycine degradation. To characterize this pathway, deletion of the gcvT homolog, an essential member of this system, was performed in attenuated and virulent F. tularensis strains. Deletion mutants were auxotrophic for serine but behaved similar to wild-type strains with respect to host cell invasion, intracellular replication, and stimulation of TNF-α. Unexpectedly, the glycine cleavage system was required for the pathogenesis of virulent F. tularensis in a murine model. Deletion of the gcvT homolog delayed mortality and lowered bacterial burden, particularly in the liver and bloodstream. To reconcile differences between the cell culture model and animal model, minimal tissue culture media was employed to mimic the nutritionally limiting environment of the host. This reevaluation demonstrated that the glycine cleavage system contributes to the intracellular replication of virulent F. tularensis in serine limiting environments. Thus, the glycine cleavage system is the serine biosynthetic pathway of F. tularensis and contributes to pathogenesis in vivo.