Epigenetic misregulation is a more common feature in human cancer than previously anticipated. In the present investigation, we identified CCCTC-binding factor (CTCF), the multivalent 11-zinc-finger nuclear factor, as a regulator that favors a particular local chromatin conformation of the human retinoblastoma gene promoter. We show that its binding contributes to Rb gene promoter epigenetic stability. Ablation of the CTCF binding site from the human Rb gene promoter induced a rapid epigenetic silencing of reporter gene expression in an integrated genome context. CTCF DNA binding is methylation sensitive, and the methylated Rb-CTCF site is recognized by the Kaiso methyl-CpG-binding protein. This is the first evidence suggesting that CTCF protects the Rb gene promoter, a classic CpG island, against DNA methylation, and when such control region is abnormally methylated Kaiso, and probably its associated repressor complex, induce epigenetic silencing of the promoter. Our results identify CTCF as a novel epigenetic regulator of the human retinoblastoma gene promoter.