The present study was conducted to investigate the effect of heat stress on antioxidative status of chicken embryo hepatocyte culture. Primary cell culture was prepared by using 14 to 16 day-old chicken embryos. Cell monolayer was incubated for 45 minutes in CO2 incubator separately at 43±0.5°C, 40±0.5°C and 37±0.50C temperature with 95% humidity and 5% CO2. After incubation, the cells were returned to 37°C temperature and allowed to recover for two hours before harvesting for preparation of cell lysate. Activity of different antioxidative enzymes and protein concentration at different incubation temperatures was estimated. Significantly (P≤0.01) lower glutathione reductase (GSH-R) and catalase (CAT) activity was recorded at 40°C and 43°C as compared to 37°C incubation temperature whereas no significant (P≥0.05) difference occurred between GSH-R and CAT activity at 40°C and 43°C. Significantly (P≤0.05) higher superoxide dismutase (SOD) activity and protein concentration was recorded at 40°C and 43°C as compared to 37°C incubation temperature whereas no significant (P≥0.05) difference occurred between SOD activity and protein concentration at 40°C and 43°C.