Aims: Anti-lipopolysaccharide factor (ALF) is an antimicrobial peptide (AMP) and a key effector molecule of the innate immune system in crustaceans. However, little is known about the role of its indirect killing against bacteria. The possible regulatory role of this peptide (M-ALF) in kuruma prawns, Marsupenaeus japonicus, was investigated. Materials and Methods: The activities of M-ALF were investigated by antimicrobial activity in vitro and by experimental infection Vibrio penaeicida in vivo with ALF-knock down in kuruma prawn by systemically silencing M-ALF gene through the injection of gene-specific long double-stranded RNA with RNA interference. Results: Synthetic M-ALF had no direct antimicrobial activity against V. penaeicida, whereas ALF-silenced kuruma prawns had significantly higher mortality than untreated prawn after V. penaeicida infection. The data provide compelling evidence that M-ALF plays an indirect protective role against V. penaeicida infection, suggesting the idea that ALF acts as a cytokine-like regulatory molecule, as well as an effector molecule. Conclusion: M-ALF has no direct activity against V. penaeicida, but might be a key molecule in cytokine-like gene regulation in crustaceans.