Enhanced virulence or infectivity after passage through a mammalian host has been reported for a number of enteric food-borne pathogens. Citrobacter rodentium is a mouse pathogen that mimics many aspects of enterohemorrhagic Escherichia coli infection of humans and serves as a useful model for studying virulence mechanisms. Emergence of a hyperinfectious state after passage through mouse gastrointestinal tract was reported for C. rodentium. We wanted to investigate if increased acid tolerance could explain hypervirulence status of C. rodentium. Although we were able to observe hyperinfectious state of C. rodentium upon host passage, the cells were extremely acid sensitive. Growth under mildly acidic conditions (LB-MES, pH 5.5) induced acid tolerance of C. rodentium, but did not improve the organism's ability to establish infection. Growth under anaerobic environment on fecal components also did not induce hyperinfectious state. Thus, contrary to conventional anticipation, hypervirulent C. rodentium cells were found to be acid sensitive thereby revealing limitations of the role of mouse gastric acidity by itself in elucidating the hypervirulent phenotype.