Megalocytivirus is a severe pathogen to turbot (Scophthalmus maximus), a popular aquaculture species in many countries. In this study, we investigated the effect of temperature on the antiviral response of turbot at transcriptome level. We found that when turbot were infected with megalocytivirus RBIV-C1 at low temperatures (14 °C, 16 °C, and 18 °C), viral replication was undetectable or moderate and no fish mortality occurred; in contrast, when turbot were infected with RBIV-C1 at high temperatures (20 °C, 22 °C, and 24 °C), viral replication was robust and 100% host mortality was observed. During the course of viral infection, downward temperature shift curbed viral replication and augmented host survival, whereas upward temperature shift promoted viral replication and reduced host survival. Comparative transcriptome analyses were conducted to examine the whole-genome transcription of turbot infected with RBIV-C1 at 16 °C and 22 °C for 4 days (samples S16-4d and S22-4d, respectively) and 8 days (samples S16-8d and S22-8d, respectively). The results showed that compared to S22-4d and S22-8d, 1600 and 5927 upregulated unigenes of various functional categories were identified in S16-4d and S16-8d, respectively. Of these genes, 22 were immune-related, most of which were detected in S16-8d and exhibited more genetic subtypes in S16-8d than in S16-4d. In addition, upregulated genes associated with cell junctions and cell membrane were also identified. These results indicate that temperature had a profound effect on the global transcription of turbot, which consequently affects the immune as well as physical resistance of the fish against viral infection.