Animal welfare and production are negatively affected by high ambient temperature. Although the gastrointestinal tract is extremely heat-sensitive, there is a dearth of information pertaining to how such heat stress affects different intestinal regions in chickens. This study therefore aimed to evaluate the morphological changes and gene expression of duodenal toll-like receptor 4 (TLR4) signaling in broilers that have been exposed to heat stress conditions on a controlled diet. At 2, 4, and 6 h of heat challenge, chickens were slaughtered, and duodenum samples were collected. Morphological changes were determined by hematoxylin and eosin staining. Five genes, TLR4, myeloid differentiation primary response gene 88 (MyD88), nuclear factor kappa B (NF-κB), toll-interleukin receptor-domain-containing adapter-inducing interferon beta (TRIF), and interferon regulatory factor 7 (IRF7), were selected to monitor the direction of the TLR4 signaling pathway. Results indicated that the duodenal intima was damaged by heat stress, TLR4 significantly responded to the heat stress, and increased heat challenge had no considerable impact on gene expression. Heat stress significantly increased MyD88 and NF-κB in MyD88-dependent signaling pathways. In conclusion, our results demonstrated that heat stress damages duodenal endothelium and upregulates duodenal TLR4–MYD88–NF-κB signaling genes in broilers.