Understanding mechanisms controlling expression of the α-spectrin gene is important for understanding erythropoiesis, membrane biogenesis, and spectrin-linked hemolytic anemia. We showed previously that a minimal α-spectrin promoter directed low levels of expression only in early erythroid development, indicating elements outside the promoter are required for expression in adult erythrocytes. Addition of noncoding exon 1′ and intron 1′ conferred a 10-fold increase in activity in reporter gene assays. In this report, we used a transgenic mouse model to show that addition of exon 1′ and intron 1′ to the α-spectrin promoter conferred tissue-specific expression of a linked ^Aγ-globin gene in erythroid cells at all developmental stages. Expression was nearly position-independent, as 21 of 23 lines expressed the transgene, and γ-globin protein was present in 100% of erythrocytes, indicating uniform expression. Additional in vivo studies revealed that exon 1′ functions as an insulator with barrier-element activity. Chromatin immunoprecipitation assays demonstrated that this region was occupied by the upstream stimulatory factors 1/2 (USF1/USF2), similar to the well-characterized chicken HS4 insulator. These data identify the first barrier element described in an erythrocyte membrane protein gene and indicate that exon 1′ and intron 1′ are excellent candidate regions for mutations in patients with spectrin-linked hemolytic anemia.