Ovine Mx1 (oMx1) is expressed in the uterus during the estrous cycle and is strongly up-regulated during early pregnancy in the uterus and peripheral blood leukocytes. Mx proteins are GTPases which are important elements in innate immunity. Our results showed that steroids are required for oMx1 protein induction by IFN in the uterus. To addresses the role of IFN in regulating oMx1 expression, a 2.1 kb fragment containing 1.7 kb of the promoter/enhancer, exon1 and partial intron A was cloned. Serial deletions were prepared along with a clone that contained the 2 promoter-ISREs (-101 & −145) but not the intronic-ISRE (+224). An ovine uterine cell line was transfected with reporter plasmids driven by the oMx1 promoter deletion constructs. The full-length promoter was induced by IFN in a dose- and time-dependent manner. Treatment with 10,000 AVU/mL IFN increased luciferase activity 5- and 10-fold at 3 and 12 hr, respectively. Promoter deletions showed the 2 proximal ISRE (−101 and −145), but not an intronic ISRE (+244), were required for maximal response. Deletion of a distal region (−920 to −715) resulted in decreased luciferase activity (~4-fold). However, subsequent deletion of the −715 to −437 region restored maximal promoter response (~10-fold). Results suggest that regions −920 to −715 and −715 to −437 have positive and negative regulatory element binding sites, respectively. The importance of the 2 proximal ISRE sites for oMx1 promoter activation is consistent with results for the human MxA promoter. An intronic ISRE is present in the human MxA gene, however, this site may not be required for oMx1 promoter activation by IFN. Identifying positive and negative regulatory regions in oMx1 promoter may help elucidate the unique regulation of Mx1 during early pregnancy.