Hirame rhabdovirus (HIRRV) is a rhabdovirus that causes severe disease in fish. The mortality due to HIRRV infection occurs at temperatures below 15 °C, but no mortality is observed over 20 °C. In this study, Japanese flounder (Paralichthys olivaceus) was artificially infected with the HIRRV CNPo2015 strain at 10 °C or 20 °C. Absolute quantitative real-time PCR was employed to examine the viral replication in spleens after HIRRV infection. Expression profiles of four interferon-related genes (type I IFN, Mx, ISG15, MDA5) and two proinflammatory genes (TNF-α and IL-1β) were also investigated by quantitative real-time PCR. Results showed that viral copies in spleens increased gradually over time and peaked at 72 h post infection (hpi) in the 10 °C group, while viral copies in the 20 °C group increased within 24 hpi, but afterwards decreased to very low levels. Moreover, the expressions of IFNs in the 10 °C group reached the highest levels at 72 hpi, whereas their peak levels appeared much earlier in the 20 °C group, at 12 hpi. The expression levels of TNF-α and IL-1β in the 10 °C group peaked at 12 hpi and then quickly declined. However, the two genes were highly expressed during 6–24 hpi in the 20 °C group. Based on these findings, we concluded that HIRRV infection induced an efficient antiviral immune response at 20 °C, which might inhibit the viral transcription at early stages and finally prevent HIRRV infection.