A viral hemorrhagic septicemia virus (VHSV), recombinant glycoprotein vaccine (VHSVg) and a DNA vaccine (pCMV-VHSg) were injected in Japanese flounder ( Paralichthys olivaceus). Each fish was injected with 10 μg vaccine dissolved in 50 μl phosphate buffer saline (PBS). One month after the vaccination, the fish were challenged intraperitoneally with either 1 × 10 2 or 1 × 10 3 TCID 50 of virus. Fish that received the VHSg DNA vaccine were highly protected against virus infection over a 21-day observation period, with cumulative mortalities ranging from 4 to 10%. However, the recombinant protein vaccine group appeared to have low survival rates. Using microarray analysis, humoral defense-related genes such as complement component C3, complement regulatory plasma proteins, IgM, IgD, MHC class II-associated invariant chain and CD20 receptor were observed to be up-regulated by the VHSg recombinant protein vaccine at 1 or 21 days post vaccination. On the other hand, cellular defense-related genes such as CD8 alpha chain, T-cell immune regulator, MIP1-α and apoptosis-associated protein were not detected. Specific antibodies against VHSVg protein were detected in both vaccinated groups at a titer of 1:40 at 28 days post vaccination.