The human ankyrin-1 gene (ANK1) contains 3 tissue-specific alternative promoters. We have shown previously that the erythroid-specific ankyrin 1 (ANK1E) core promoter contains a 5' DNase I hypersensitive site (HS) with barrier insulator function that prevents gene silencing in vitro and in vivo. Mutations in the ANK1E barrier region lead to decreased ANK1 mRNA levels and hereditary spherocytosis. In this report, we demonstrate a second ANK1E regulatory element located in an adjacent pair of DNase I HS located 5.6 kb 3' of the ANK1E promoter at the 3' boundary of an erythroid-specific DNase I-sensitive chromatin domain. The 3' regulatory element exhibits enhancer activity in vitro and in transgenic mice, and it has the histone modifications associated with an enhancer element. One of the ANK1E 3'HS contains an NF-E2 binding site that is required for enhancer function. We show that a chromatin loop brings the 3' enhancer and NF-E2 into proximity with the 5' barrier region including the ANK1E core promoter. These observations demonstrate a model for the tissue-specific activation of alternative promoters that may be applicable to the ∼ 30% of mammalian genes with alternative promoters that exhibit distinct expression patterns.