Increasing temperature and exercise disrupt tight junctions of the gastrointestinal tract although the contribution of environmental temperature to intestinal damage when exercising is unknown. This study investigated the effect of 2 different environmental temperatures on intestinal damage when exercising at the same relative intensity. Twelve men (mean ± SD; body mass, 81.98 ± 7.95 kg; height, 182.6 ± 7.4 cm) completed randomised cycling trials (45 min, 70% maximal oxygen uptake) in 30 °C/40% relative humidity (RH) and 20 °C/40%RH. A subset of participants (n = 5) also completed a seated passive trial (30 °C/40%RH). Rectal temperature and thermal sensation (TSS) were recorded during each trial and venous blood samples collected at pre- and post-trial for the analysis of intestinal fatty acid-binding protein (I-FABP) level as a marker of intestinal damage. Oxygen uptake was similar between 30 °C and 20 °C exercise trials, as intended (p = 0.94). I-FABP increased after exercise at 30 °C (pre-exercise: 585 ± 188 pg·mL−1; postexercise: 954 ± 411 pg·mL−1) and 20 °C (pre-exercise: 571 ± 175 pg·mL−1; postexercise: 852 ± 317 pg·mL−1) (p < 0.0001) but the magnitude of damage was similar between temperatures (p = 0.58). There was no significant increase in I-FABP concentration following passive heat exposure (p = 0.59). Rectal temperature increased during exercise trials (p < 0.001), but not the passive trial (p = 0.084). TSS increased more when exercising in 30 °C compared with 20 °C (p < 0.001). There was an increase in TSS during the passive heat trial (p = 0.03). Intestinal damage, as measured by I-FABP, following exercise in the heat was similar to when exercising in a cooler environment at the same relative intensity. Passive heat exposure did not increase I-FABP. It is suggested that when exercising in conditions of compensable heat stress, the increase in intestinal damage is predominantly attributable to the exercise component, rather than environmental conditions.