Exertional heat stress can cause damage to the intestinal epithelium and disrupt gastrointestinal barrier integrity, leading to microbial translocation (MT) linked to the development of heat stroke. This study aimed to assess age-related differences in markers of intestinal epithelial injury and MT following non-heat stress and high-heat stress exercise in healthy young and older men. Markers of intestinal epithelial injury (intestinal fatty acid-binding protein-'IFABP') and MT (soluble cluster of differentiation 14-'sCD14'; and lipopolysaccharide-binding protein-'LBP') were assessed in healthy young (18-30 y, n = 13) and older (50-70 y) men (n = 12). Blood samples were collected before, after 180min of moderate-intensity (metabolic rate: 200 W/m2) walking and following 60min recovery in either a non-heat stress [temperate: 21.9°C, 35% relative humidity (RH)] or high-heat stress (hot: 41.4°C, 35% RH) environment. There were no differences in IFABP and sCD14 between the young and older groups in the temperate condition, while LBP was greater in the older group (+ 0.66 ug/mL; + 0.08 to + 1.24 ug/mL). In the hot condition, the older group experienced greater increases in IFABP compared to the young group (+ 712pg/mL/hr; + 269 to + 1154pg/mL/hr). However, there were no clear between-group differences for sCD14 (+ 0.24 ug/mL/hr, - 0.22 to + 0.70 ug/mL/hr) or LBP (+ 0.86 ug/mL/hr, - 0.73 to + 2.46 ug/mL/hr). While older men may experience greater intestinal epithelial injury following exercise in the heat; this did not lead to a greater magnitude of microbial translocation relative to their younger counterparts.