CTCF is a key regulator of gene expression through organization of the chromatin structure. Still, it is unclear how CTCF binding is perturbed in leukemia or in cancer in general. We studied CTCF binding by ChIP-Seq in cells from patients with acute myeloid leukemia (AML) and in normal bone marrow (NBM) in the context of gene expression, DNA methylation and azacytidine exposure. CTCF binding was increased in AML compared to NBM. Aberrant CTCF binding was enriched for motifs for key myeloid transcription factors such as CEBPA, PU.1 and RUNX1. AML with TET2 mutations was characterized by a particularly strong gain of CTCF binding, highly enriched for gain in promoter regions while AML in general was enriched for changes at enhancers. There was a strong anti-correlation between CTCF binding and DNA methylation. Gain of CTCF occupancy was associated with increased gene expression, however, the genomic location (promoter vs. distal regions) and enrichment of motifs (for repressing vs. activating co-factors) were decisive for the gene expression pattern. Knock-down of CTCF in K562 cells caused loss of CTCF binding and transcriptional repression of genes with changed CTCF binding in AML, as well as loss of RUNX1 binding at RUNX1/CTCF binding sites. In addition, CTCF knock-down caused increased differentiation. Azacytidine exposure caused major changes in CTCF occupancy in AML patient cells, partly by restoring a CTCF binding pattern similar to NBM. We conclude that AML displays an aberrant increase in CTCF occupancy that targets key genes for AML development and impacts on gene expression.