Abstract Background Anastomotic leakage (AL) is the most dreaded complication after colorectal surgery, causing high morbidity and mortality. Mucus is a first line of defence against external factors in the gastrointestinal tract. In this study, the structural mucus protein Muc2 was depleted in genetically engineered mice and the effect on healing of colonic anastomoses studied in an experimental model. Methods Mice of different Muc2 genotypes were used in a proximal colonic AL model. Tissues were scored histologically for inflammation, bacterial translocation was determined by quantitative PCR of bacterial 16S ribosomal DNA, and epithelial cell damage was determined by assessing serum levels of intestinal fatty acid-binding protein. Results Of 22 Muc2-deficient (Muc2−/−) mice, 20 developed AL, compared with seven of 22 control animals (P < 0·001). Control mice showed normal healing, whereas Muc2−/− mice had more inflammation with less collagen deposition and neoangiogenesis. A tendency towards higher bacterial translocation was seen in mesenteric lymph nodes and spleen in Muc2−/− mice. Intestinal fatty acid-binding protein levels were significantly higher in Muc2−/− mice compared with controls (P = 0·011). Conclusion A functional mucous layer facilitates the healing of colonic anastomoses.