Background Intestinal failure–associated liver disease causes significant mortality in patients with short bowel syndrome. Steatosis, a major component of intestinal failure–associated liver disease has been shown to persist even after weaning from parenteral nutrition. We sought to determine whether steatosis occurs in our murine model of short bowel syndrome and whether steatosis was affected by manipulation of the intestinal microbiome. Methods Male C57BL6 mice underwent 50% small bowel resection and orogastric gavage with vancomycin or vehicle for 10 weeks. DNA was extracted from stool samples then sequenced using 16s rRNA. Liver lipid content was analyzed. Bile acids were measured in liver and stool. Results Compared with unoperated mice, small bowel resection resulted in significant changes in the fecal microbiome and was associated with a >25-fold increase in steatosis. Oral vancomycin profoundly altered the gut microbiome and was associated with a 15-fold reduction in hepatic lipid content after resection. There was a 17-fold reduction in fecal secondary bile acids after vancomycin treatment. Conclusion Massive small bowel resection in mice is associated with development of steatosis and prevented by oral vancomycin. These findings implicate a critical role for gut bacteria in intestinal failure–associated liver disease pathogenesis and illuminate a novel, operative model for future investigation into this important morbidity.