Several regulatory regions are important for the expression of genes encoding T helper type 2 (T_H2) cytokines, including T_H2-specific DNase I hypersensitivity sites in the T_H2 cytokine locus control region. Among these sites, Rad50 hypersensitive site 7 (RHS7) shows rapid T_H2-specific demethylation after antigenic stimulation. To investigate the function of RHS7 in T_H2 cell differentiation, we have generated RHS7-deficient mice. CD4⁺ T cells and mast cells showed a notable reduction in T_H2 cytokine expression in vitro and T_H2 responses in vivo were considerably impaired in RHS7-deficient mice. Deletion of RHS7 did not affect the expression of a linked Rad50 gene, but it did reduce long-range intrachromosomal interactions between the locus control region and promoters of the T_H2 cytokine genes. Our findings show that RHS7 is essential for the proper regulation of T_H2 cytokine gene expression.