In this study, we examined the protein and gene expression of heat shock proteins (HSPs) in different sections of the small intestine of chickens. In total, 300 one-day-old Ross 308 broiler chicks were randomly allocated to the control and treatment groups. The treatment group was divided into four subgroups, according to the duration of acute heat exposure (3, 6, 12, and 24 h). The influence of heat stress on the protein and gene expression of HSP70, HSP60, and HSP47 in different sections of the small intestine of chickens was determined. The protein expression of HSP70 and HSP60 was significantly higher at 6 h in the duodenum and jejunum and 12 h in the ileum. The HSP47 protein expression was significantly higher at 3 h in the duodenum and ileum and at 6 h in the jejunum. The gene expression levels of HSP70, HSP60, and HSP47 were significantly higher at the 3 h treatment group than the control group in the duodenum, jejunum, and ileum. The glutamate pyruvate transaminase and glutamate oxaloacetate transaminase levels were significantly higher at 12 and 24 h in the serum of the blood. Acute heat stress affected the expression of intestinal proteins and genes in chickens, until the induction of heat tolerance.