Cohesins, which mediate sister chromatid cohesion and are essential for chromosome segregation and DNA repair, also regulate gene expression and enhancer-promoter interactions. The mechanisms underlying these non-canonical functions have been unclear due to a lack of knowledge about cohesin-binding sites and interactors in metazoans. This study reveals that cohesins in mammalian cells occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling those of CTCF, a DNA-binding protein with enhancer-blocking and boundary-element activities. CTCF is required for cohesin localization to these sites, suggesting a functional relationship between the two proteins. The sequence motif enriched among mammalian cohesin sites is highly similar to the CTCF consensus sequence, and cohesin recruitment by CTCF explains the existence of cell-specific cohesin sites. This mechanism integrates DNA sequence and epigenetic state, providing a rationale for the non-canonical functions of cohesins.Cohesins, which mediate sister chromatid cohesion and are essential for chromosome segregation and DNA repair, also regulate gene expression and enhancer-promoter interactions. The mechanisms underlying these non-canonical functions have been unclear due to a lack of knowledge about cohesin-binding sites and interactors in metazoans. This study reveals that cohesins in mammalian cells occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling those of CTCF, a DNA-binding protein with enhancer-blocking and boundary-element activities. CTCF is required for cohesin localization to these sites, suggesting a functional relationship between the two proteins. The sequence motif enriched among mammalian cohesin sites is highly similar to the CTCF consensus sequence, and cohesin recruitment by CTCF explains the existence of cell-specific cohesin sites. This mechanism integrates DNA sequence and epigenetic state, providing a rationale for the non-canonical functions of cohesins.