Recent studies have demonstrated that the synthetic human defensin-alpha1, also designated as human neutrophil peptide 1 (HNP1), not only has in vitro antiviral activity against viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, but can also modulate some immune activities of rainbow trout (Oncorhynchus mykiss) head kidney leucocytes. However, none of these HNP1 properties have been analysed in vivo so far. Thus, in the current work, we have studied the in vivo immunomodulatory capacity of HNP1 on the rainbow trout immune system as a first approach to evaluate the possible use of this family of antimicrobial peptides (AMPs) to increase fish resistance by enhancing non-specific defence mechanisms. The intramuscular injection of synthetic HNP1 induced the transcript expression of genes encoding both pro-inflammatory cytokines (IL-1beta, TNF-alpha1 and specially IL-8) and CC chemokines (CK5B, CK6 and CK7A) as well as of the genes related to type I interferon (IFN) production (Mx1, Mx2, Mx3 and IFN regulatory factor 3, IRF-3) in different trout tissues (muscle, head kidney and blood). Furthermore, the chemotactic capacity of HNP1 towards trout leucocytes has been clearly revealed. All together, these results demonstrate that in vivo HNP1 is active across species and can modulate fish immune responses. Therefore, in a moment when most pathogens have developed resistance to commonly used antibiotics, natural antimicrobial peptides with inter-specific activity, such as HNP1, might prove to be useful model molecules for the development of novel therapeutic agents that exhibit both microbicidal and immunoenhancing capabilities.