Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type β- or γ-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type β-globin expression cassette into the human genome for sustained expression of β-globin. We initially constructed a β-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken β-actin promoter (CAGGS) and full-length β-globin cDNA, as well as truncated forms lacking either the 3′ or 3′ and 5′ untranslated regions (UTRs), to optimize expression of β-globin. β-Globin with its 5′ UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis 573-Tn-β-globin plasmids using a minimal β-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human αLCR, ankyrin-1 promoter), IHβp (human ALAS2 intron 8 crythroid-specific enhancer, HS40 core element from human αLCR, β-globin promoter), or HS3βp (HS3 core element from human βLCR, β-globin promoter) to establish erythroid-specific expression of β-globin. Stable genomic insertion of the minimal gene and expression of the β-globin transgene for >5 months at a level comparable to that of the endogenous γ-lobin gene were achieved using a SB-Tn β-globin cis construct. Interestingly, erythroid-specific expression of β-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of β-globin.