Exposure to high temperature is an inherent feature of grass carp culture in southern China and juvenile grass carps are predisposed to infectious disease in this condition. To understand how high temperature impacts the immune response to pathogens in grass carp, the transcriptomic profiles of the spleens from immune injected grass carp groups undergoing heat stress and normal temperature were investigated. An average of 72 million clean reads per library was obtained, and approximate 80% of these genes were successfully mapped to the reference genome. A total of 2287 up-regulated and 1068 down-regulated genes were identified. 10 immune-related categories involving 90 differently expressed genes were scrutinized. Expression patterns of 18 differentially expressed genes involving in immune response were validated by quantitative real-time RT-PCR. These results provide further significant insights into the influence mechanism of high temperature to immune response in grass carp.