show Abstracthide AbstractChromatin organization is critical for cell growth, differentiation, and disease development, however, its functions in peripheral myelination and myelin repair remain elusive. Here we observed a global diminution of chromatin accessibility during Schwann cell differentiation and demonstrated that the chromatin organizer CCCTC-binding factor (CTCF) is critical for Schwann cell myelination and myelin regeneration after nerve injury. Inhibition of Ctcf or its deletion blocked Schwann cell differentiation at the pre-myelinating stage, whereas overexpression of CTCF promoted the myelination program. CTCF establishes the chromatin interaction loop between promoters and regulatory elements to promote expression of key pro-myelinogenic factors such as EGR2. In addition, CTCF interacts with SUZ12, a component of polycomb-repressive-complex 2, to repress expression of immature Schwann cell-associated regulators including HES1, RSPO2, and CALCA. Together, our findings reveal the dual role of CTCF-dependent chromatin organization in promoting myelinogenic programs and recruiting chromatin-repressive complexes to block differentiation inhibitors to control peripheral myelination and myelin repair. Overall design: RNA-seq: 2 Control and 2 Ctcf cKO mouse sciatic nerves; 3 siControl and 3 siCtcf rat Schwann cell cultures. ChIP-seq: 1 proliferating and 1 differentiating samples for CTCF ChIP-seq; 1 siControl and 1 siCtcf samples for H3K27ac ChIP-seq. ATAC-seq: 2 siControl and 2 siCtcf samples.