Neutrophil recruitment to the injured tissues appears to be an evolutionarily conserved strategy for organisms to fight against exogenous insults.Recent studies show rhythmic migration of neutrophils and that several factors including melatonin have been implicated in regulating this rhythmic migration.The mechanisms underlying how endogenous melatonin regulates rhythmic neutrophil migration,however,are unclear.Here we generated a zebrafish annat2 mutant that lacks endogenous melatonin and Tg(lyz:EGFP);aanat2-/-transgenic line that allows for monitoring neutrophil migration visually in live zebrafish.We observed that migrating neutrophils are significantly reduced in aanat2 mutant zebrafish under light/dark condition,and disrupted migrating rhythmicity of neutrophils in aanat2-/-zebrafish is independent of the circadian clock.Further,we also found that endogenous melatonin enhances neutrophil migration likely through inducing expression of cytokines such as Interleukin-8 and Interleukin-1β.Together,our findings provide the evidence that endogenous melatonin promotes rhythmic migration of neutrophils through cytokines in zebrafish.