Innate and acquired immune responses provide critical line in defense against pathogens. MicroRNAs (miRNAs) play crucial roles in regulate gene expression in inflammation and innate immunity. However, the main role of miRNAs are in a large part unclear in aquatic organisms. In this study, we found that miR-122 displayed dramatically declined expression profiles in Vibrio anguillarum challenged miiuy croaker. Meanwhile, we have explored TLR14 as a novel target gene of miR-122 involved in miiuy croakers inflammatory and immune response, which were further estimated through negative expression profiles in both Vibrio anguillarum challenged miiuy croaker and LPS exposure macrophages. Finally, in the dual-luciferase reporter assay presented mmi-miR-122 negatively regulated the 3′-UTR of wild-type in luciferase activity rather than the mutant one in HEK-293T cells. This result demonstrated that mmi-miR-122 modulated TLR14 expression by directly targeting TLR14–3′UTR. All of the present data suggested miR-122 was involved in TLR cascade could modulate Vibrio anguillarum infection in miiuy croakers.