Citrobacter rodentium belongs to a family of human and animal enteric pathogens that includes the clinically significant enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens use attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract. In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity, dynamics of colonization and clearance of mice by C. rodentium in situ and in real time. The bioluminescent C. rodentium derivative, strain ICC180, expresses the luxCDABE operon from the entemopathogenic nematode symbiont Photorhabdus luminescens and light levels accurately reflect bacterial numbers both in vitro and in vivo. We have demonstrated that primary colonization of the mouse by C. rodentium takes place within the caecum, specifically within the specialized patch of lymphoid tissue known as the caecal patch. Following colonization of the caecum C. rodentium established a colonic infection. Clearance of C. rodentium ICC180 parallels the colonization dynamics, i.e. the caecum was first to be cleared followed by the colon. A bioluminescent eae (encoding the outer membrane adhesin intimin) C. rodentium mutant failed to establish long-term colonization, although low levels of bacteria could be recovered for up to 3 days post challenge from the caecum.