Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia in fish, particularly in channel catfish. Ferric iron is an essential micronutrient for bacterial survival, and some bacterial pathogens use secreted hydroxamate-type siderophores to chelate iron in host tissues. Siderophore-iron complexes are taken up by these bacteria via the ferric hydroxamate uptake (Fhu) system. In E. ictaluri, the Fhu system consists of fhuC, fhuD, fhuB, and fhuA genes. However, the importance of the Fhu system in E. ictaluri virulence has not been investigated completely. Here, we present construction of E. ictaluri fhuD and fhuB mutants (EiΔfhuD and EiΔfhuB) by in-frame gene deletion and evaluation of the mutants' virulence and immunogenicity in channel catfish fingerlings and fry. Immersion challenges showed that EiΔfhuD was not significantly attenuated (p < 0.05) in catfish fingerlings, whereas EiΔfhuB was significantly attenuated (p < 0.01). Catfish fingerlings immunized with EiΔfhuD and EiΔfhuB showed 100% and 97.62% survival, respectively. Fry immersion challenges indicated EiΔfhuB was also significantly attenuated (p < 0.05) in two-week old fry compared to the wild-type (48.96% vs. 82.14% mortalities). The survival rate in the fry vaccinated with EiΔfhuB was significantly higher (p < 0.05) than that of non-vaccinated fry (96.77% vs. 21.42% survival). Our data indicates that the fhuB gene, but not the fhuD gene, contributes to E. ictaluri virulence.