Human β-thalassemia is closely associated with aberrant expression of β-like globin genes. Human β-like globin genes are organized in the order of 5′-ε-Gγ-Aγ-δ-β-3′ within the β-globin locus. The expression of β-like globin genes is regulated by 3′HS1 and five DNase I hypersensitive sites (5′HS5~5′HS1) in a locus control region. The 5′HS2 enhancer transcribes enhancer RNA and regulates the expression of ε-globin, γ-globin and β-globin. To further study the function of 5′HS2, we detected the local 3D genomic architecture via chromatin conformation capture experiments and used CRISPR/ Cas9-based DNA fragment editing to delete 5′HS2 in human K562 leukaemia cells. In this study, we found that 5′HS2-mediated chromatin interactions were enriched in a topologically associated domain that was bordered by 3′HS1 and 5′HS5. Within this topologically associated domain, 5′HS2 is highly close to the promoter regions of HBE1, HBG2 and HBG1. Upon deletion of the 5′HS2 enhancer, 91 genes were significantly down-regulated with reduced abundance of H3K27ac at their promoter regions. These down-regulated genes are mainly associated with oxygen transport, immune response, cell adhesion, anti-oxidant and thrombosis. These data suggested that many genes associated with functions of erythrocytes were decreased at transcriptional levels upon deletion of the 5′HS2 enhancer.