Formalin killed Pseudomonas anguilliseptica bacterial vaccine was prepared and administered to farm reared olive flounder, Paralichthys olivaceus reared at 17 °C and 20 °C for 4 weeks. Non-vaccinated fishes (n=150) served as positive control. Vaccinated fishes were divided into two groups (n=150 each in replicate). Both the vaccinated and non-vaccinated fishes were challenged intraperitoneally with P. anguilliseptica (1×10(7) CFU ml(-1)) isolates and PBS (negative control). Fishes were sampled from zero hour post injection (hpi) for 28 days (each hour and each day); the mean percent mortality and relative percent survival (RPS) were calculated for the challenged and control groups. The vaccinated fishes had a significant increase in RPS (69 and 89, respectively); the percentage mortality declined from 83±0.6 and 74±0.7 in challenged and control fishes to 25%±0.8% and 8%±0.8% in vaccinated and challenged fish groups, respectively. The immune gene expression assay was analyzed using real-time PCR. Vaccinated fishes registered a significant increase in the expression of TNFR-1, FasL, IRF7, TLR2, IL-1b and CD40 gene transcripts when compared to the control group. The upregulation of these genes along with the increased RPS values suggest that the formalin-killed cells of P. anguilliseptica could play an important role in immunizing olive flounder against P. anguilliseptica.