Previous studies demonstrated the occurrence of bacterial translocation in biliary obstruction (BO). In this study, we investigated the effect of lactulose on the prevention of bacterial translocation in rats with BO. The study was performed in 4 groups of rats consisting of 15 animals each: (1) sham-operated controls, (2) sham-operated lactulose-treated, (3) BO physiologic-saline-treated and (4) BO lactulose-treated rats. Lactulose was given perorally by orogastric intubation in a dose of 2 ml of 33.5% solution/day. Fourteen days after BO, bacterial translocation to the mesenteric lymph nodes (MLN), liver, spleen and portal venous blood (PVB) was investigated. In sham-operated controls, 1 bacterial translocation to the PVB was observed. In the BO physiologic-saline-treated group, the rate of bacterial translocation to the MLN (p < 0.001) and the PVB (p < 0.001) was significantly higher as compared with sham-operated controls and the BO lactulose-treated group. Lactulose treatment caused a significant decrease in the number of gram-negative aerobes and facultative anaerobes (p < 0.001) and a significant increase in the number of lactobacilli (p < 0.05) in the cecal population levels of bacteria in sham-operated and BO groups as compared to sham-operated controls and BO saline-treated groups. In histopathological examinations of the terminal ileum and the cecum, subepithelial edema and mucosal disruption were observed in the BO saline-treated group. Lactulose treatment significantly decreased sub-epithelial edema in the terminal ileum (p = 0.02) and the cecum (p = 0.04), and the surface epithelium was normal in rats with BO treated with lactulose compared to the BO physiologic-saline-treated group. Lactulose treatment significantly increased the mucosal thickness in the terminal ileum and cecum as compared to the nontreated groups (p < 0.01 and p < 0.0001). It is concluded that peroral treatment with lactulose significantly reduced the rate of bacterial translocation to the MLN and PVB in rats with BO.