The Type I interferons are critical mediators of antimicrobial function and cell turnover. Increasingly, IFN has been demonstrated to have a vital role in the maintenance of intestinal homeostasis and protection from inflammatory bowel disease (IBD). RNase-L is a downstream mediator of IFN function that also induces a positive feedback mechanism through innate immune receptors to amplify IFN levels. RNase-L is widely accepted as an antiviral protein and its antibacterial functions have recently been elucidated. The goal of this work was to investigate the role of RNase-L in gastrointestinal (GI) diseases to discover new therapeutic targets with translational potential. First, the role of RNase-L in experimental colitis and colitis-associated cancer was investigated. Next, the role of IFN expression and RNase-L activity following infection with a diarrheal agent that causes mortality in infants, Enteropathogenic Escherichia coli (EPEC), was determined. Vital roles for IFN and RNase-L in intestinal inflammatory disease and infection were identified. Results demonstrated that RNase-L is protective against injury-induced colitis through regulation of the immune response including increased IFN expression, and that RNase-L inhibits tumor growth in experimental colitis-associated cancer (CAC). Antibacterial function against EPEC by IFN and RNase-L were also demonstrated. IFN signaling was found to have a critical role in intestinal epithelial cell (IEC) homeostasis and protection against EPEC infection. Furthermore, RNase-L was necessary to prevent translocation of EPEC across the IEC barrier. Supporting a protective role for IFN and RNase-L, secreted EPEC effectors inhibited IFN expression and RNase-L activity. Results indicate that IFN and RNase-L are important mediators of intestinal homeostasis with protective roles against infection and inflammatory disease