In the present study, juvenile rainbow trout ( Oncorhynchus mykiss) were acclimated at 5 temperatures. Fish in the control group (C 0) were reared at a constant temperature (16°C); trout in four other treatments (A 0, A 3, A 6, and A 9) were acclimated to a high temperature (22°C) for 0, 3, 6, and 9 d, respectively, and then returned to normal temperature (16°C) for 7 d. The temperature was then raised to 20°C and the fish were cultured for 40 d. The results showed that the growth rates of A 3, A 6, and A 9 were higher than that of A 0 but lower than that of C 0. The growth rate of A 9 was the highest among the 4 acclimation groups. The activities of serum transaminase and liver antioxidant enzyme significantly increased during acclimation. At the beginning of growth, the activities of enzymes were lower in A 3, A 6, and A 9 with the lowest in A 9. In addition, the transcript abundance of heat shock protein (HSP) 60 gene in A 9 was not significantly different from that of C 0 during growth. HSP70 in A 9 significantly increased at the beginning and returned to that of C 0 at the end. Our findings indicated that pre-acclimation improved the high temperature tolerance with the best effectiveness observed at 22°C for 9 d. A possible mechanism underlining such phenomenon is the improvement of antioxidant defense system.