To determine the effects of heat acclimation on gastrointestinal (GI) damage and the gastric emptying (GE) rate following endurance exercise in a hot environment. Fifteen healthy men were divided into two groups: endurance training in hot (HOT, 35 °C, n = 8) or cool (COOL, 18 °C, n = 7) environment. All subjects completed 10 days of endurance training (eight sessions of 60 min continuous exercise at 50% of the maximal oxygen uptake (V·O2max). Subjects completed a heat stress exercise tests (HST, 60 min exercise at 60% V·O2max) to evaluate the plasma intestinal fatty acid-binding protein (I-FABP) level and the GE rate following endurance exercise in a hot environment (35 °C) before (pre-HST) and after (post-HST) the training period. We assessed the GE rate using the 13C-sodium acetate breath test. The core temperature during post-HST exercise decreased significantly in the HOT group compared to the pre-HST (p = 0.004) but not in the COOL group. Both the HOT and COOL groups showed exercise-induced plasma I-FABP elevations in the pre-HST (p = 0.002). Both groups had significantly attenuated exercise-induced I-FABP elevation in the post-HST. However, the reduction of exercise-induced I-FABP elevation was not different significantly between both groups. GE rate following HST did not change between pre- and post-HST in both groups, with no significant difference between two groups in the post-HST. Ten days of endurance training in a hot environment improved thermoregulation, whereas exercise-induced GI damage and delay of GE rate were not further attenuated compared with training in a cool environment.