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Innate immunity is initiated depending on the recognition of certain protein receptors termed pattern recognition receptors (PRRs) of pathogen-associated molecular patterns (PAMPs) to protect the host from various invading pathogens. As one of the most powerful PAMPs, flagellin is the major structural component of the flagellum that provides the main force for bacterial motility in flagellated microorganisms. The genome of the Y. ruckeri strain SC09 contains three flagellin genes, which encode the flagellins FlaA, FlaB and FlaC, respectively. In this study, we produced the three full-length recombinant flagellins—i.e., rFlaA, rFlaB and rFlaC—from the Y. ruckeri strain SC09 for the first time and then compared the host cell responses to rFlaA, rFlaB and rFlaC using channel catfish cultured head kidney monocytes/macrophages in vitro. Moreover, the time-dependent modulation of the nine genes expression of primary kidneys injected with rFlaC was also detected by qPCR. We found that rFlaA, rFlaB and rFlaC all can stimulate the production of some pro-inflammatory cytokines, such as IL1-β1, TNFα, IL8, iNOS1 and Hepcidin. In addition, the expression of TLR5M, TLR5S, NF-κB and MHC II β was all increased after channel catfish cultured head kidney monocytes/macrophages were stimulated by the three recombinant flagellins. Importantly, rFlaC stimulated the highest expression of all the genes mentioned above compared with that of rFlaB and rFlaA and enhanced the expression of the nine above-mentioned genes in vivo. Our study lays the foundation for the effect of flagellin on immune responses, suggesting that flagellin may be a useful immune adjuvant or stimulant in the aquaculture field.Three recombinant flagellins from Y. ruckeri were obtained.Host cell responses to rFlaA, rFlaB and rFlaC were compared in vitro.rFlaC was a more optimal immune adjuvant than rFlaA and/or rFlaB in aquaculture field.