This study investigated the effects of fish oil (FO) supplementation on the hypothalamus heat resistance gene expressions and fatty acid composition of chicks under acute high-temperature stress, for treating Cholestasis. A total of 48 chicks (Ross 308) at age of 14 days were acclimatized to corn oil or FO (n = 24 for each) by oral gavaging for 10 days, and then subjected to heat stress (35 ± 1 °C, HT) for 3 h or maintained at the normal temperature (26 ± 1 °C, NT) as grouped as NT and FO-NT control and HT and FO-HT (n = 12 for each).. The results showed that FO supplementation had no significant (P >
0.05) effect on feed intake or body weight. The FO-HT group exhibited (P<
0.05) a lower rectal temperature, and plasma interleukin-6 (IL-6), triglyceride and corticosterone levels, in accompany with lower expressions of hypothalamic adenine nucleotide translocators (ANT) and uncoupling protein (UCP) but increased (P<
0.05) plasma superoxide dismutase activity and hypothalamic neuropeptide-Y (NPY) and heat shock protein-70 (HSP-70) expressions.. Additionally, the FO-HT group (P <
0.05) demonstrated a higher unsaturated fatty acid/saturated fatty acid (UFA/SFA) ratio in the breast muscle. These findings suggest that FO supplementation can enhance the heat resistance of broiler chicks under acute heat stress and alter the fatty acid composition of their breast muscle. However, further studies are needed to determine whether desirable fatty acids can cross the blood-brain barrier and their implications for human health.