CTCF (CCCTC-binding factor) is a highly conserved multifunctional DNA-binding protein with thousands of binding sites genome-wide. Our previous work suggested that differences in CTCF's binding site sequence may affect the regulation of CTCF recruitment and its function. To investigate this possibility, we characterized changes in genome-wide CTCF binding and gene expression during differentiation of mouse embryonic stem cells. After separating CTCF sites into three classes (LowOc, MedOc and HighOc) based on similarity to the consensus motif, we found that developmentally regulated CTCF binding occurs preferentially at LowOc sites, which have lower similarity to the consensus. By measuring the affinity of CTCF for selected sites, we show that sites lost during differentiation are enriched in motifs associated with weaker CTCF binding in vitro. Specifically, enrichment for T at the 18(th) position of the CTCF binding site is associated with regulated binding in the LowOc class and can predictably reduce CTCF affinity for binding sites. Finally, by comparing changes in CTCF binding with changes in gene expression during differentiation, we show that LowOc and HighOc sites are associated with distinct regulatory functions. Our results suggest that the regulatory control of CTCF is dependent in part on specific motifs within its binding site.