The current study was conducted to investigate the impact of thermal stress on growth performance, blood biomarkers, metabolic hormones, immunological response, antioxidant activity, and expression of stress-related genes in broilers. One hundred and fifty one-day-old chicks (Ross 308) were utilized in this work. On the 21st day of age, birds were subjected to three environmentally controlled treatments with five replicate pens of 10 birds per each, representing an initial density of 10 birds/m 2 -control: reared in a thermoneutral condition; THS 1 and THS 2: exposed to 4 and 6 h of daily thermal stress at 40 ± 1°C, respectively, until the 42 days of age. The results demonstrated that thermal stress for 4 and 6 h significantly reduced (P < 0.001) daily weight gain, daily feed intake, blood leukocyte and lymphocyte counts, serum immunoglobulins (IgM, IgA, IgG), and insulin-like growth factor-1 (IGF-1), while serum levels of aspartate aminotransferase, alanine aminotransferase, glucose, cholesterol, low-density lipoprotein, and lactate dehydrogenase were elevated relative to the thermoneutral group. Additionally, the corticosterone level and the ratio of heterophil:lymphocyte increased significantly (P < 0.001) in thermal-stressed groups. The antioxidant enzymes were affected by thermal stress as represented by a significant decrease in the activity of serum catalase (CAT) and glutathione peroxidase (GSH-Px) along with an increase in malonaldehyde concentration. Thermal stress affected gene expression by upregulating heat shock protein 70, heat shock factors 1 and 3, nuclear factor kappa B, interleukin-4, and uncoupling protein, and downregulating GSH-Px, CAT, and IGF-1 transcript levels. However, no changes were observed in interleukin-2 expression levels. It can be concluded that thermal stress destructively influences productivity, physiological status, and gene expression by upregulating heat shock protein 70, heat shock factors 1 and 3, nuclear factor kappa B, interleukin-4, and uncoupling protein, and downregulating GSH-Px, CAT, and IGF-1 transcript levels of broiler chickens.