This research was executed to study the impacts of adding betaine (BT) to broiler diets on intestinal inflammatory response and barrier integrity under heat stress (HS). At 21 d of age, 150 male broilers (Ross 308) were randomly assigned to 3 treatment groups: control (CON) group, in which broilers were provided standard finisher feed under thermoneutral condition (22 ± 1°C); HS group and HS + BT group, in which broilers were given the standard feed supplied with 0 and 1,000 mg/kg BT, respectively, under cyclic HS condition (33 ± 1°C for 8 h from 08:00 to 16:00 h and the thermoneutral temperature for the residual hours). Each treatment was replicated ten times with 5 broilers per replicate. The HS group showed an elevation (P < 0.05) in serum corticosterone (CORT) concentration, D-lactate acid (D-LA) content, and diamine oxidase (DAO) activity, mucosal interleukin-1β (IL-1β) level, and expression of heat shock protein 70 (HSP70) gene, and a reduction (P < 0.05) in mucosal interleukin-10 (IL-10) level and secretory immunoglobulin A (SIgA) content and relative abundance of mRNA for occludin (OCLN), zonula occludens-1 (ZO-1), claudin-1 (CLDN1), and claudin-4 (CLDN4). In contrast, broilers in the HS + BT group exhibited a raise (P < 0.05) in mucosal IL-10 level and SIgA content and relative expression of OCLN and ZO-1 genes, and a decline (P < 0.05) in serum CORT concentration and DAO activity, mucosal IL-1β level, and expression of HSP70 mRNA. These results indicate that supplemental BT can ameliorate intestinal injury in heat-challenged broilers by suppressing inflammatory responses and enhancing mucosal barrier function.