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Vibrio anguillarum and Edwardsiella tarda are severe aquaculture pathogens shared similar epidemiological characteristics and susceptible to flounder ( Paralichthys olivaceus) . In our previous studies, recombinant(r) protein heat shock protein 33 (rHsp33) from V. anguillarum and outer membrane protein C (rOmpC) from E. tarda were proved to have protection against V. anguillarum and E. tarda , respectively. In this paper, the cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum , and the protection of divalent vaccine candidate (rHsp33 + rOmpC, rHC) against both V. anguillarum and E. tarda were evaluated. RHC, rHsp33, and rOmpC were vaccinated to flounder, respectively, and the percentages of surface immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBLs), serum IgM, specific antibodies against V. anguillarum or E. tarda , specific antibodies against rHsp33, rOmpC or rHC, the expression of immune-related genes and relative percent survival (RPS) against V. anguillarum or E. tarda were measured. The results showed that: RHC could induced the enhancement of sIg + cells and high levels of specific antibodies against both V. anguillarm and E. tarda ; Also a significant increase of specific antibodies against rHsp33, rOmpC or rHC, and up-regulation of gene expression of CD3, CD4-1, CD4-2, CD8α, CD8β and IgM in spleen, head-kidney, and hindgut, RPS of 70 ± 3.45% against V. anguillarum and 60 ± 1.48% against E. tarda , respectively. In addition, rHsp33 induced specific antibodies against E. tarda and rOmpC, and had a RPS of 43.3 ± 3.73% against E. tarda ; rOmpC could evoke specific antibodies against V. anguillarum and rHsp33, and had a RPS of 44 ± 1.27% against V. anguillarm ; The results demonstrated that there was cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum , rHC as a divalent vaccine can induce significant immune response and efficient protection against both E. tarda and V. anguillarum in flounder. [ABSTRACT FROM AUTHOR]