Background: Bovine Mx2 gene sequences were already reported, but further information about the gene properties is not yet available. The objective of the current study was to elucidate the structural properties of the bovine Mx2 gene mainly the promoter region and its possible functional role. If available, such information would help in assessing the functional properties of the gene, which was reported to confer antiviral action against recombinant VSV. Results: Examinations on the bovine genomic BAC clone—confirmed to contain the Mx2 gene—revealed 883-bp sequences. A computer scan unequivocally identified a 788-bp promoter region containing a typical TATA box, three ISREs and other promoter-specific motifs. Comparative analysis of nine bovine genomic DNA samples showed 19 nucleotide substitutions suggesting the existence of five different genotypes in the promoter region. The water buffalo Mx2 promoter region was determined by using primers based on the bovine Mx2 promoter region disclosing 893-bp, with 56 substitutions, two insertions, 9 and 1 nt at two different sites. A functional analysis of the putative ISRE indicated that ISRE played a synergetic role in the activation of bovine Mx2 gene transcription. Conclusion: Bovine and water buffalo Mx2 promoter region was identified disclosing, the conserved ISRE, located in the proximal end of the promoter region like other members of the antiviral family, suggesting functional activity under interferon stimulation.