CTCF binds to the consensus sequence CCGCGNGGNGGCAG (in IUPAC notation). This sequence is defined by 11 zinc finger motifs in its structure. CTCF's binding is disrupted by CpG methylation of the DNA it binds to. On the other hand, CTCF binding may set boundaries for the spreading of DNA methylation. In recent studies, CTCF binding loss is reported to increase localized CpG methylation, which reflected another epigenetic remodeling role of CTCF in human genome.
CTCF binds to an average of about 55,000 DNA sites in 19 diverse cell types (12 normal and 7 immortal) and in total 77,811 distinct binding sites across all 19 cell types.
CTCF's ability to bind to multiple sequences through the usage of various combinations of its zinc fingers earned it the status of a “multivalent protein”.  More than 30,000 CTCF binding sites have been characterized. The human genome contains anywhere between 15,000 and 40,000 CTCF binding sites depending on cell type, suggesting a widespread role for CTCF in gene regulation. In addition CTCF binding sites act as nucleosome positioning anchors so that, when used to align various genomic signals, multiple flanking nucleosomes can be readily identified. On the other hand, high-resolution nucleosome mapping studies have demonstrated that the differences of CTCF binding between cell types may be attributed to the differences in nucleosome locations. Methylation loss at CTCF-binding site of some genes has been found to be related to human diseases, including male infertility.