Using ENU mutagenesis two mouse strains called Winnie and Eeyore were generated with a single missense mutation in a N- terminal and C-terminal Muc2 D-domain. These mice have a phenotype similar to human ulcerative colitis (UC), including smaller and fewer goblet cells, decreased production of mucus, chronic diarrhoea and intestinal inflammation. Muc2 mucin is the major component of intestinal mucus, it contains N- and C-terminal D-domains involved in homo-oligomerisation. We sought to determine how a missense mutation in the Muc2 mucin gene initiates an UC-like phenotype in mice. Histological analysis of Winnie and Eeyore mice showed they had fewer goblet cells with smaller theca, increased intestinal permeability, increased proliferation and apoptosis, as well as abnormal accumulations of non-O-glycosylated Muc2 surrounding the nucleus of goblet cells. The regions of Muc2 accumulation were shown to contain dilated endoplasmic reticulum (ER) indicating that Muc2 was misfolding and accumulating within the ER which is a classic sign of ER stress. To further examine Muc2 misfolding, two N-terminal regions which both contained the Winnie mutation were cloned and expressed in vitro. These N-terminal proteins containing the Winnie mutation were shown to hyper-oligomerise and were blocked within the secretory pathway in MKN45 cells. This provides evidence that the Winnie mutation causes Muc2 to misfold and accumulate within cells. In order to assess the susceptibility of Winnie mice to infection they were infected with bacterial pathogens and assessed for their level of infection. After 9 and 14 days of infection with the intestinal bacterial pathogen Citrobacter rodentium, Winnie mice showed significantly higher levels of intestinal and systemic infection than wild type mice. However, even though Winnie mice had higher levels of infection, they were able to clear the infection before wild type mice, possibly due to pre-existing inflammation and earlier/more vigorous engagement of adaptive immunity. We conclude the Winnie and Eeyore mutations result in inappropriate oligomerisation and accumulation of a proportion of Muc2 in the ER, causing ER stress. ER stress in goblet cells leads to increased apoptosis and inflammation, as well as less secreted mucin which can result in a reduced ability to exclude pathogens. Together with evidence that ER stress occurs in goblet cells in UC, these data suggest that ER stress may have a pivotal role in UC pathophysiology.