Many viral vaccines used in aquaculture are unable to stimulate an appropriate level of immunity to withstand infection. By targeting specific components of the immune system it may be possible to trigger stronger, more effective responses to antigens. Flagellin has the ability to stimulate both the soluble and membrane-bound forms of toll-like receptor 5 (TLR5) in salmon leading to a proinflammatory response and activation of both the innate and adaptive immune system. In this study flagellin (FlaD from Vibrio anguillarum) was recombinantly produced in two forms, full-length (FDL) and a truncated form (FDS) with portions of the N- and C-termini removed to prevent polymerization. FDS was used to produce an antibody that was able to bind both forms of flagellin in immunoblot analysis. In cell culture using COS-7 cells, FDL was shown to stimulate the NF-κB pathway more effectively than FDS. Both forms of flagellin were used as an adjuvant with the antigen LPH (Hemocyanin from Limulus polyphemus hemolymph) in an immunization dose-response study. FDS and FDL stimulated the innate immune system of salmon inducing proinflammatory effects on days 2, 4 and 7 and the gene expression of important cytokines such as TNFα, IL-6, IL-8, and IL-1β were significantly up-regulated (p<0.05) in the spleen. TLR5S was more highly up-regulated than TLR5M indicating that the soluble form of TLR5 may play an important role in the innate immune response in salmon. ELISA analysis showed that the use of flagellin as an adjuvant with LPH was not able to significantly induce flagellin or LPH antibodies. This study shows that flagellin has the potential to be a highly effective adjuvant for salmon immunization, but further research is needed.