Biliary tract infection (BTI)-derived sepsis remains a serious problem with significant morbidity and mortality in the modern era of critical care management. Current animal models of BTI have relied mostly on injecting purified bacteria or their toxins into the biliary tract. These models do not fully reflect pathophysiology or disease processes of clinical cholangitis or cholecystitis. In the current study, we developed a novel model of BTI by performing cholecystocolonic anastomosis (CCA) in rabbits and characterized pathophysiologic changes in this model. This model is intended to mimic the clinical process of cholecystocolonic fistula with reflux cholangitis, a severe form of BTI. Adult male rabbits were subjected to BTI-derived sepsis through an anastomosis of the gall bladder to the colon (i.e., CCA). The animals were monitored for 7 days to record survival. In additional groups of animals, various bacterial, hemodynamic, histological and biochemical parameters were measured at 12, 24, 48 and 72 h after CCA. The anastomosis between the gallbladder and the colon required about 5-8 min to finish. The median survival time for rabbits after CCA was 96 h. The positive rates of bacterial culture at 72 h after CCA were 83.3% and 100% in the blood and liver, respectively. The most common microorganism was Escherichia coli followed by Enterococcus. Plasma Tumor Necrosis Factor-α (TNF-α), Lnterleukin-10 (IL-10), Lnterleukin-6 (IL-6), and High-mobility group box 1 protein (HMGB-1) levels were greatly elevated after CCA. The cardiac index and heart rate increased slightly at 12 h after CCA and then continued to decrease. Systemic hypotension developed 48 h after CCA. Histological studies showed reflux cholangitis with acute lung and kidney injury. Cholecystocolonic anastomosis produces polymicrobial sepsis in rabbits, which mimics many aspects of human BTI-derived sepsis. It is reproducible and easy to perform and may serve as an excellent model for future sepsis research.