Hepcidin acts as both an antimicrobial peptide and a hormonal regulator of iron homeostasis; however, the biological significance of this dual-function in immune reactions remains elusive. In this study, we provide experimental evidence regarding the coordination of this dual-function in the innate antimicrobial immunity using a zebrafish model. The transcription of hepcidin gene was significantly upregulated in liver by Aeromonas hydrophila (A.h) DNA stimulation, which was accompanied by an increase of hepcidin protein and a decrease of iron concentration in serum. Thus, an enhanced bactericidal activity against A.h and Escherichia coli and inhibitory effects on A.h growth and OmpA expression were observed in A.h cells, the latter of which made the bacterium more susceptible to complement attack. The enhanced bacteriostatic activities in serum following the stimulation were dramatically impaired by neutralizing hepcidin or restoring iron to the samples. Immuno-protection assay showed that zebrafish administrated with A.h DNA or designed CpG-ODNs had a significantly enhanced defence against A.h and Vibrio alginolyticus infections, which was also eliminated by the neutralization of hepcidin. Results indicate that the induction of hepcidin leads to the decrease of iron in circulation, which eventually limits iron availability to invading microorganisms, thus contributing to host defence.