It is known that thermal conditioning at an early age results in improved heat tolerance, and reduces mortality when re-exposed to heat in later life in chickens. However, the mechanism of thermal conditioning is not fully understood. The objective of this study was to investigate the effect of early thermal conditioning on physiological and behavioral responses in acute heat-exposed chicks. Six-day-old chicks (White Plymouth Rock) were exposed to high temperature at 40°C for 3 h while control chicks were kept at 30°C. Four days after treatment, both groups were challenged to high temperature at 40°C for 15 min. We found that the initiation times for behavioral responses (panting and wing-droop posture) in experienced chicks were later than those in control. At the end of heat-exposure treatment, the rectal temperature in experienced chicks was lower than that in control while there was no difference in respiration rate between the groups. Compared with control, experienced chicks had a lower level of plasma corticosterone. Gene expression levels of brain-derived neurotrophic factor, thyrotropin-releasing hormone, interleukin-6 and lipopolysaccharide-induced tumor necrosis factor were significantly lower in the brain of experienced chicks than in the control chicks. These results suggest that thermal conditioning may change response to subsequent heat exposure by altering the central thermoregulation system, resulting in an alleviation of heat stress.