Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen with an extremely low infectious dose that is responsible for major disease outbreaks. STEC colonizes the colon and can cause hemorrhagic colitis and hemolytic uremic syndrome in humans, often leading to hospitalization and sometimes resulting in death. The STEC genome contains a ure gene cluster, ureDABCEFG, which encodes the urease enzyme that produces ammonia as it hydrolyzes the substrate urea. However, the contribution of urease to the pathogenicity of STEC and the regulation of the ure gene locus had not been elucidated. Previously, in the STEC O157:H7 Sakai strain, a single nucleotide polymorphism (SNP) in ureD encoding a urease chaperone protein was identified, resulting in substitution of an amber stop codon for glutamine. This SNP is observed in the majority of STEC O157:H7 isolates and correlates with a negative urease phenotype in vitro. We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD, and experiments reveal that a L38P substitution enhances enzyme activity, however, a L205P substitution does not. Multi-locus sequence typing analysis for a variety of STEC isolates combined with ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor ureD sequence is phylogenetically restricted. Experiments conducted using a mouse model for stomach transit demonstrate that urease-expressing wild-type STEC is twice as likely to survive the acidic environment during stomach passage as the ure deletion mutant. Mouse colonization experiments also reveal an enhanced ability of the wild-type STEC to colonize the intestinal tract compared to the ure deletion mutant. Furthermore, we demonstrate that the STEC ure gene locus is nitrogen-regulated at the transcriptional level. Overall, we demonstrate that, although STEC appears to express urease at a moderate level, the ammonia produced upon urea hydrolysis most likely aids in STEC pathogenicity by buffering the bacteria during stomach transit as well as providing an easily assimilated nitrogen source that enhances colonization ability in the colon where STEC must compete with other microbes for nitrogen.