Multiple evidence connects intestinal epithelial cell endoplasmic reticulum (ER) stress with inflammation. The intestinal microbiota also plays an essential role in regulating epithelial function and intestinal inflammation. In Winnie mice, a Muc2 mutation drives ER stress in intestinal epithelial goblet cells, likely providing the initial trigger for the spontaneous colitis, with the subsequent depleted mucus barrier and increased bacterial translocation exacerbating inflammation. However, it has been difficult to ascertain the relative contributions of ER stress versus microbial translocation due to the depleted mucus barrier. This study sought to determine whether the microbiota was required for the development of Winnie spontaneous colitis, and whether inflammation affects the severity of epithelial ER stress. To address these questions we housed C57BL/6 and Winnie mice in a germ-free (GF) facility and compared the intestinal histology and molecular evidence of inflammation and ER stress with conventionally raised (CONV-R) mice, with and without immunosuppression. Spontaneous colitis was substantially reduced but not completely abolished in GF compared to CONV-R Winnie mice. Inflammatory cell infiltration was reduced but several chemokines remained high in the absence of microbes. Concomitantly, ER stress was also diminished, although mucin misfolding and mature mucin production were only partially improved. In CONV-R Winnie mice, the anti-inflammatory drug, 5-ASA, also supressed colitis and ER stress, and more effectively restored mucin biosynthesis and reduced mucin misfolding. These experiments demonstrate that epithelial intrinsic protein misfolding directly initiates inflammation resulting in chemokine release independently of microbes. However, the microbiota is necessary for the amplification of colitis in Winnie mice, resulting in production of multiple inflammatory cytokines, increased goblet cell ER stress and a diminished barrier to the microbes establishing a forward-feeding cycle of inflammation. Our results suggest that anti-inflammatory therapy could be more efficacious than targeting the microbiome for patients with genetic predispositions involving intestinal epithelial stress.