In broiler chickens, heat stress disrupts nutrient digestion and absorption. However, the underlying molecular mechanism is not clearly understood. Hence, to investigate the effects of high ambient temperatures on the expression levels of nutrient transporters in the jejunum of broiler chickens, seventy-two 35-day-old male broiler chickens with similar body weights were randomly allocated into two groups: control (24 ± 1 °C) and heat-stressed (32 ± 1 °C). The chickens in the heat-stressed group were exposed to 10 h of heat daily from 08:00 to 18:00 and then raised at 24 ± 1 °C. The rectal temperature and feed intake of the chickens were recorded daily. After 7 days, nine chickens per group were sacrificed by exsanguination, and the jejunum was collected. The results show that heat exposure significantly decreased the feed intake and increased the rectal temperature of the broiler chickens. The plasma concentrations of uric acid and triglyceride significantly increased and decreased, respectively, in the heat-stressed group. No significant differences in the levels of plasma glucose, total amino acids, and very low-density lipoprotein were observed between the heat-stressed and control groups. However, the plasma concentration of glucose tended to be higher (P = 0.09) in the heat-stressed group than in the control group. Heat exposure did not significantly affect the mRNA levels of Na+-dependent glucose transporter 1 and amino acid transporters y + LAT1, CAT1, r-BAT, and PePT-1. However, the expression levels of GLUT-2, FABP1, and CD36 were significantly decreased by heat exposure. The results of this study provide new insights into the mechanisms by which heat stress affects nutrient absorption in broiler chickens. Our findings suggest that periodic heat exposure might alter the jejunal glucose and lipid transport rather than amino acid transport. However, intestinal epithelial damage and cell loss should be considered when interpreting the effects of heat stress on