Zebrafish(Danio rerio) is an ideal model for studying the mechanism of infectious disease and the interaction between host and pathogen.As a teleost,zebrafish has developed a complete immune system which is similar to mammals.Moreover,the easy acquirement of large amounts of transparent embryos makes it a good candidate for gene manipulation and drug screening.In a zebrafish infection model,all of the site,timing,and dose of the bacteria microinjection into the embryo are important factors that determine the bacterial infection of host.Here,we established a multi-site infection model in zebrafish larvae of 36 hours post-fertilization(hpf) by micro-injecting wild-type or GFP-expressing Staphylococcus aereus(5.aureus) with gradient burdens into different embryo sites including the pericardial cavity(PC),eye,the fourth hindbrain ventricle(4V),yolk circulation valley(YCV),caudal vein(CV),yolk body(YB),and Duct of Cuvier(DC) to resemble human infectious disease.With the combination of GFP-expressing S.aureus and transgenic zebrafish Tg(corola:eGFP;lyz:Dsred) and Tg(lyz:Dsred) lines whose macrophages or neutrophils are fluorescent labeled,we observed the dynamic process of bacterial infection by in vivo multicolored confocal fluorescence imaging.Analyses of zebrafish embryo survival, bacterial proliferation and myeloid cells phagocytosis show that the site- and dose-dependent differences exist in infection of different bacterial entry routes.This work provides a consideration for the future study of pathogenesis and host resistance through selection of multi-site infection model.More interaction mechanisms between pathogenic bacteria virulence factors and the immune responses of zebrafish could be determined through zebrafish multi-site infection model. Zebrafish (Danio rerio) is an ideal model for studying the mechanism of infectious disease and the interaction between host and pathogen. As a teleost, zebrafish has developed a complete immune system which is similar to mammals. More over, the easy acquirement of large amounts of transparent embryos makes it a good candidate for gene manipulation and drug screening. a of zebrafish infection model, all of the site, timing, and dose of the bacteria microinjection into the embryo are important factors that determine the bacterial infection of host. Here, we established a multi-site infection model in zebrafish larvae of 36 hours post-fertilization (hpf) by micro-injecting wild-type or GFP-expressing Staphylococcus aereus (5.aureus) with gradient burdens into different embryo sites including the pericardial cavity ), the fourth hindbrain ventricle (4V), the yolk circulation valley (YCV), the caudal vein (CV), the yolk body (YB), and the Duct of Cuvier (DC) to resemble human infectious disease. e xpressing S. aureus and transgenic zebrafish Tg (corola: eGFP; lyz: Dsred) and Tg (lyz: Dsred) lines whose macrophages or neutrophils are fluorescent labeled, we observed the dynamic process of bacterial infection by in vivo multicolored confocal fluorescence imaging. Analyses of zebrafish embryo survival, bacterial proliferation and myeloid cells phagocytosis show that the site- and dose-dependent differences exist in infection of different bacterial entry routes. This work provides a consideration for the future study of pathogenesis and host resistance through selection of multi-site infection model. Minor interactions mechanisms between pathogenic bacteria virulence factors and the immune responses of zebrafish could be determined through zebrafish multi-site infection model.