Antimicrobial peptides (AMPs) play essential roles in the innate immune system to protect against a wide variety of pathogens in aquatic environments. In this study, three very important AMPs, cathelicidin, hepcidin and defensin, were identified in the critically endangered Acipenser dabryanus. The full-length cDNA sequences of these three AMPs were identified from transcriptome sequencing and the rapid amplification of cDNA ends (RACE) technique. Phylogenetic analysis showed that cathelicidin formed a clade with the other members of the cathelicidin family, and similar results were obtained for hepcidin. The A. dabryanus β-defensin belonged to the fish class 2 β-defensins. A tissue distribution study showed that the three AMP transcripts could be detected constitutively in various tissues. The highest expression levels of cathelicidin and hepcidin were found in the liver, while defensin was primarily expressed in the skin. Bacterial challenge in vivo revealed significant changes in the gene expression of the three AMPs at both mucosal sites and systemic sites. Striking upregulation of cathelicidin and hepcidin was observed in the skin at 12 h post-challenge, with increases of more than 7000-fold and 1000-fold, respectively, compared to the control, and the expression of defensin mRNA was remarkably elevated in the hindgut (by 230-fold at 6 h post-challenge). Moreover, according to the expression profiles of the AMPs post-challenge, we found that the mucosal immune response occurred earlier than the systemic immune response following bacterial infection. Our results suggest that these three novel AMPs may play important roles in the innate immune system of A. dabryanus to protect against invading pathogens, especially during the mucosal immune response.