Purpose: Exertional-heat stress adversely disrupts gastrointestinal (GI) barrier integrity and, through subsequent microbial translocation (MT), can have negative health consequences for physically active populations (e.g. military personnel, athletes, laborers). Acute glutamine (GLN) supplementation is a potential nutritional countermeasure (Zuhl et al., 2015; Pugh et al., 2017), although doses previously validated for this purpose can cause symptoms of GI intolerance (e.g. nausea, bloating; Ward et al., 2003).  Method: Ten moderately trained males completed two 80-minute exertional-heat stress tests (EHST) separated by 7-14 days with a double-blind, randomised, counterbalanced, cross-over design. Low dose oral GLN (0.3 g·kg-1 fat free mass) or PLA beverages were ingested one hour before commencing the EHST. Venous blood was drawn immediately pre- and post- EHST. GI barrier integrity was assessed using the serum dual-sugar absorption test (DSAT) and plasma Intestinal Fatty-Acid Binding Protein (I-FABP). MT was assessed using plasma total 16S bacterial DNA and Bacteroides/total 16S DNA. Results: Whole-body physiological and perceptual strain were comparable between the GLN and PLA trials during the EHST (p ˃ 0.05). The GLN bolus was well tolerated, with no evidence of GI intolerance. Serum DSAT responses were similar (p = 0.14) between the GLN (0.029 ± 0.023) and PLA trials (0.023 ± 0.005), though the post-EHST elevation in I-FABP was more pronounced with GLN (Δ = 2.542 ± 1.205 ng·ml-1) than PLA (Δ= 1.374 ± 1.101 ng·ml-1) supplementation (p = 0.01). Bacteroides/total 16S DNA responses tended to increase (p = 0.06) following the EHST, but there was no difference between the GLN (Δ = 0.02 ± 0.05) and PLA (Δ = 0.16 ± 0.34) trials. Conclusion: Acute low-dose (0.3 g·kg-1 fat free mass) oral GLN supplementation one hour before exertional-heat stress enhances I-FABP concentrations, but this does not translate to augmented GI MT.