Urease represents a critical virulence factor for some bacteria species through its alkalizing effect that helps neutralize the acidic microenvironment of the pathogen. In addition, urease serves as a nitrogen source provider for bacterial growth. Pathogenic mycobacteria express a functional urease but its role during infection has yet to be characterized. Here, we constructed a urease-deficient M. tuberculosis (Mtb) strain and confirmed the alkalizing effect of the urease activity within the mycobacteria-containing vacuole in resting macrophages but not in the more acidic phagolysosomal compartment of activated macrophages. However, the urease-mediated alkalizing effect did not confer any growth advantage to Mtb in macrophage as evidenced by comparable growth profiles for the mutant, wild type (WT) and complemented strains. In contrast, the urease-deficient mutant exhibited impaired in vitro growth compared to the WT and complemented strains when urea is the sole source of nitrogen. Substantial amounts of ammonia were produced by the WT and complemented strains, but not with the urease-deficient mutant, which represents the actual nitrogen source for mycobacterial growth. However, the urease-deficient mutant displayed parental colonization profiles in the lungs, spleen and liver in mice. Together, our data demonstrate a role of the urease activity in Mtb nitrogen metabolism that could be crucial for the pathogen's survival in nutrient-limited microenvironments where urea is the sole nitrogen source. Our work supports the notion that Mtb virulence correlates with its unique metabolic versatility and ability to utilize virtually any carbon and nitrogen sources available in its environment