Spatial expression patterns of homeobox(HOX) genes delineate positional identity of primary fibroblasts from different topographic sites. The molecular mechanism underlying the establishing or maintaining of HOX gene expression pattern remains an attractive developmental issue to be addressed. Our previous work suggested a critical role of CTCF/cohesin-mediated higher-order chromatin structure in RA-induced HOXA activation in human teratocarcinoma NT2/D1 cells. This study investigated the recruitment of CTCF and cohesin, and the higher-order chromatin structure of the HOXA locus in fetal lung and adult foreskin fibroblasts, which display complementary HOXA gene expression patterns. Chromatin contacts between the CTCF-binding sites were observed with lower frequency in human foreskin fibroblasts. This observation is consistent with the lower level of cohesin recruitment and 5′ HOXA gene expression in the same cells. We also showed that CTCF-binding site A56(CBSA56) related chromatin structures exhibit the most notable changes in between the two types of cell, and hence may stand for one of the key CTCF-binding sites for cell-type specific chromatin structure organization. Together, these results imply that CTCF/cohesin coordinates HOXA cluster higher-order chromatin structure and expression during development, and provide insight into the relationship between cell-type specific chromatin organization and the spatial collinearity. Spatial expression patterns of homeobox (HOX) genes delineate positional identity of primary fibroblasts from different topographic sites. The molecular mechanism underlying the establishing or maintaining of HOX gene expression patterns remains an attractive developmental issue to be addressed. Our previous work suggested a critical role of CTCF / cohesin-mediated higher-order chromatin structure in RA-induced HOXA activation in human teratocarcinoma NT2 / D1 cells. This study investigated the recruitment of CTCF and cohesin, and the higher-order chromatin structure of the HOXA locus in fetal lung and adult foreskin fibroblasts, which display complementary HOXA gene expression patterns. Chromatin contacts between the CTCF-binding sites were observed with lower frequency in human foreskin fibroblasts. This observation is consistent with the lower level of cohesin recruitment and 5 ’HOXA gene expression in the same cells We also showed that CTCF-binding site A56 (CBSA56) related chromatin s tructures exhibit the most notable changes in between the two types of cell, and hence may stand for one of the key CTCF-binding sites for cell-type specific chromatin structure organizations. Together, these results imply that CTCF / cohesin coordinates HOXA cluster higher- order chromatin structure and expression during development, and provide insight into the relationship between cell-type specific chromatin organization and the spatial collinearity.