Mx proteins are one of the most studied interferon-stimulated genes (ISGs). The antiviral activity against different fish viruses has been demonstrated for diverse fish Mx proteins, including the Senegalese sole (Solea senegalensis) Mx protein (SsMx). The aim of the current study is to characterize the structure and functional activity of the SsMx promoter. Several polyclonal cell populations expressing the luciferase reporter gene under the control of the SsMx promoter have been used to determine the ability of this promoter to drive the expression of the luciferase gene after poly I:C stimulation. In addition, the implication of each interferon-stimulated response element (ISRE) in the activation of the promoter has also been analyzed. The genomic structure of the Senegalese sole and Japanese flounder Mx promoters (containing three ISREs) differs from the rest of the fish Mx promoters described to date. The ISRE1, the one closest to the start codon, is the main ISRE involved in the SsMx promoter activity, whereas ISRE2 and ISRE3 show a minor additive effect on this activity. Another feature differing SsMx promoter from the rest of the fish Mx promoters is the presence of a 24-bp GC island close to the ATG codon, including one Sp1 binding site, which may constitute the transcriptional start site. Furthermore, the SsMx promoter contains a gamma interferon activation site (GAS) element.