The management of incubation conditions impacts embryonic development, hatchability, and post-hatch performance. This study aimed to examine the effects of thermal manipulation (TM) during embryonic development on roosters' thermotolerance, antioxidant activity, immunity, and semen quality under heat-stress conditions. 1200 fertile eggs were distributed evenly between two groups, each containing three replicates (200 eggs/replicate). The first group (G1) was held in the commercial setter with a consistent temperature of 37.5°C and 55% relative humidity (RH) through the 18-day incubation period, acting as a control, while the second group (G2) experienced these conditions until only the 11th day. The eggs were incubated at 39.5°C with 60% RH for 4 h each day from the 12th to the 18th day. From the 19th to 22nd incubation days, both groups maintained a consistent temperature of 37.2°C with a RH of 70%. Two hundred hatched male chicks per treatment group were moved into a closed-system house. All roosters were exposed to a 6-h daily heat challenge with a temperature of 35°C and a humidity of 70% between their 36th and 40th weeks of age. Roosters of G2 exposed to thermal challenge showed improvements (p ≤ 0.05) in multiple blood biochemical, antioxidant, and immunity markers, including total protein, globulin, aspartate aminotransferase, alanine aminotransferase, triiodothyronine, thyroxine, corticosterone, testosterone, total antioxidant capacity, malondialdehyde, immunoglobulin G, immunoglobulin M, and immunoglobulin A levels. Improved semen quality characteristics, including ejaculate volume, sperm concentration, motility, livability, and quality factor, as well as enhanced thermoregulation in post-hatch cocks, were also achieved (p ≤ 0.05). To boost antioxidant activity, immunity, thermotolerance, and semen parameters in roosters under heat-stress conditions, TM application during egg incubation, specifically at 12-18 days, is recommended.