The protective effects of a subunit vaccine, a recombinant protein of Edwardsiella tarda glyceraldehyde 3-phosphate dehydrogenase (Rec) combined with peptidoglycan (PGN) and polyinosinic:polycytidylic acid (polyIC), against edwardsiellosis in Japanese flounder Paralichthys olivaceus were investigated to evaluate an adjuvant property of pathogen-associated molecular patterns. By injection of the mixture of the recombinant protein plus peptidoglycan and polyIC (Rec + PGN and Rec + IC, respectively), the interleukin-1β and interferon (IFN)-γ mRNA levels were upregulated in comparison to injection of phosphate-buffered saline (PBS). In addition, type I IFN mRNA was induced in the Rec + IC group. Although antibody titers were increased not only in the recombinant injected Rec group but also in the Rec + PGN and Rec + IC groups, the Rec + PGN and Rec + IC values were slightly higher and lower than those of the Rec group, respectively. After the challenge test with E. tarda, the Rec + IC group showed a higher survival rate than the PBS group, whereas the rates of the Rec and Rec + PGN groups were lower than the PBS group. Furthermore, bacteria were reisolated from only 1 out of 16 survivors in the Rec + IC group. These results suggested that polyIC might be a candidate for a vaccine adjuvant for intracellular bacterial infection.