microRNAs (miRNAs) are highly conserved, small non-coding endogenous molecule, and can participate in a variety of biological processes in organisms such as development, growth and immune response. Dual-Specificity Phosphatases (DUSPs) are enzymes that can remove phosphate groups from phosphatases. Research found that DUSP1 is an important molecule in the process of MAPK regulation. However, as a significant regulatory factor, the study of DUSP1 was very few in fish. Consequently, in this study, the regulatory role of miRNAs on DUSP1 has been verified through dual-luciferase reporter assay and western blotting analysis. Furethermore, we found that miR-375 mimics and pre-miR-375 plasmid can negatively regulate the target gene DUSP1 in miiuy croaker through combining with 3’untranslated region of DUSP1 gene. These experiment results directly indicate the negative regulatory function of miR-375 to DUSP1. Moreover, miR-375 can negatively regulate NF-κB signaling pathway via target to DUSP1. This study can increase our knowledge and help us to understand complexity of genomic and complex gene expression regulatory networks in teleost fish.