The role of nitric oxide (NO) in inflammatory bowel diseases has traditionally focused on the inducible form of NO synthase (iNOS). However, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms may also impact on colitis, either by contributing to the inflammation, or by regulating mucosal integrity in response to noxious stimuli. To date, studies examining the roles of the NOS isoforms in experimental colitis have been conflicting, and the mechanisms by which these enzymes exert their effects remain unclear. To investigate and clarify the roles of the NOS isoforms in gut inflammation, we induced TNBS colitis in eNOS, nNOS and iNOS-knockout (ko) mice, assessing the course of colitis at early and late times. Both eNOS and iNOS ko mice developed a more severe colitis compared to wildtype mice. During colitis; iNOS expression dramatically increased on epithelial and lamina propria mononuclear cells while eNOS expression remained localized to endothelial cells. Electron and fluorescence microscopy identified bacteria in the ulcerated colonic mucosa of eNOS ko mice, but not in wildtype, iNOS or nNOS ko mice. Further, eNOS ko mice had fewer colonic goblet cells, impaired mucin production and exhibited increased susceptibility to an inflammatory stimulus that was sub- threshold to other mice. This susceptibility was reversible since the NO donor isosorbide dinitrate normalized goblet cell numbers and ameliorated subsequent colitis in eNOS ko mice. These results identify a protective role for both iNOS and eNOS during colitis, with eNOS deficiency resulting in impaired intestinal defense against lumenal bacteria and increased susceptibility to colitis.