Yersinia ruckeri is known to cause enteric red mouth disease (ERM) in channel catfish (Ictalurus punctatus). This study established a model of Y. ruckeri-induced intestinal inflammation in channel catfish. Subsequently, using quantitative polymerase chain reaction (qPCR), gene cloning, recombinant protein expression, protein molecular docking, and tissue pathology techniques, we investigated the role and molecular mechanism of Flagellin C (FliC) from Y. ruckeri in inducing inflammation. The findings indicated that FliC was the main virulence gene in Y. ruckeri responsible for inducing intestinal inflammation. Specifically, FliC bound to the host Toll-like receptor 5 (tlr5), leading to the upregulation of multiple inflammatory factors such as tumor necrosis factor (tnf)-α, interleukin (il)-6, and il-1β, and the activation of the nuclear factor-kappaB (NF-κB) and JAK-STAT signaling pathways, thereby initiating inflammation. The results were validated through experiments conducted both in cellular models and in vivo. In summary, this study identified FliC as a virulence gene in Y. ruckeri infection of channel catfish and elucidated its role in inducing intestinal inflammation.