The first step in nosocomial infections due to Klebsiella pneumoniae is colonization of the patient's gastrointestinal (GI) tract. In a previous work, signature-tagged mutagenesis was used in a murine model to identify 13 genes required for efficient colonization, two of which were involved in urea metabolism. The role of urease was further investigated by the construction and analysis of an isogenic urease-deficient mutant. The behavior of both the wild-type strain and the urease-deficient mutant was tested under hostile conditions, reproducing stresses encountered in the GI environment. The wild-type strain had an acid tolerance response (ATR) to inorganic acid, was resistant to organic acids (38.5% survival) and was able to survive concentrations of bile encountered in vivo. The absence of urease did not affect the resistance of K. pneumoniae to acid and bile stresses, but the enhanced adhesion response to Int-407 cells after exposure to bile observed with the wild-type strain was no longer detected with the urease mutant. When tested in the murine intestinal colonization model, both strains were mainly recovered in the large intestine parts, and the mutant was impaired in its colonization capacities, but only when tested in competition with the wild-type strain. These findings emphasize the prominent role played by metabolic function in the colonization process of such a complex ecosystem as the host GI tract.