Background: Short bowel syndrome (SBS) is associated with gut barrier dysfunction. We examined effects of dietary glutamine (GLN) or oral antibiotics on indices of gut barrier function in a rat model of SBS. Methods: Adult rats underwent a 60% distal small bowel + proximal colonic resection (RX) or bowel transection (TX; control). Rats were pair-fed diets, with or without L-GLN for 20 days after operation. Oral antibiotics [neomycin, metronidazole, and polymyxin B; (ABX)] were given in some RX rats fed control diet. Stool secretory immunoglobulin A (sIgA) was measured serially. On day 21, mesenteric lymph nodes (MLN) were cultured for gram-negative bacteria. IgA-positive plasma cells in jejunum, stool levels of flagellin- and lipopolysaccharide (LPS)-specific sIgA and serum anti-flagellin- and anti-LPS IgG levels were determined. Results: RX caused gram-negative bacterial translocation to MLN, increased serum total and anti-LPS IgG and increased stool total sIgA. After RX, dietary GLN tended to blunt bacterial translocation to MLN (-29%, P =NS) and significantly decreased anti-LPS IgG levels in serum, increased both stool and jejunal mucosal sIgA, and increased stool anti-LPS-specific IgA. Oral ABX eliminated RX-induced bacterial translocation, significantly decreased total and anti-LPS IgG levels in serum, significantly decreased stool total IgA, and increased stool LPS-specific IgA. Conclusions: Partial small bowel-colonic resection in rats is associated with gram-negative bacterial translocation from the gut and a concomitant adaptive immune response to LPS. These indices of gut barrier dysfunction are ameliorated or blunted by administration of dietary GLN or oral antibiotics, respectively. Dietary GLN upregulates small bowel sIgA in this model.