Enterococcus faecalis is an intestinal commensal that cannot synthesize porphyrins and only expresses a functional respiratory chain when provided with exogenous haematin. In the absence of haematin, E. faecalis reverts to fermentative metabolism and produces extracellular superoxide that can damage epithelial-cell DNA. The acute response of the colonic mucosa to haematin-starved E. faecalis was identified by gene array. E. faecalis was inoculated into murine colons using a surgical ligation model that preserved tissue architecture and homeostasis. The mucosa was exposed to haematin-starved E. faecalis and compared with a control consisting of the same strain grown with haematin. At 1 h post-inoculation, 6 mucosal genes were differentially regulated and this increased to 42 genes at 6 h. At 6 h, a highly significant biological interaction network was identified with functions that included nuclear factor-kappaB (NF-kappaB) signalling, apoptosis and cell-cycle regulation. Colon biopsies showed no histological abnormalities by haematoxylin and eosin staining. Immunohistochemical staining, however, detected NF-kappaB activation in tissue macrophages using antibodies to the nuclear localization sequence for p65 and the F4/80 marker for murine macrophages. Similarly, haematin-starved E. faecalis strongly activated NF-kappaB in murine macrophages in vitro. Furthermore, primary and transformed colonic epithelial cells activated the G2/M checkpoint in vitro following exposure to haematin-starved E. faecalis. Modulation of this cell-cycle checkpoint was due to extracellular superoxide produced as a result of the respiratory block in haematin-starved E. faecalis. These results demonstrate that the uniquely dichotomous metabolism of E. faecalis can significantly modulate gene expression in the colonic mucosa for pathways associated with inflammation, apoptosis and cell-cycle regulation.