Igk VJ rearrangement occurs only in one allele. It is not fully understood how this allelic exclusion is regulated. We previously identified a recombination silencer and heterochromatin targeting element in the mouse Igk gene locus, which is termed Sis. Using chromosome conformation capture (3C) technology, here, we demonstrate that Sis and Igk gene intronic enhancer (Ei) exhibits physical interaction with the intervening DNA looping out. Knock-down of CCCTC-binding factor, CTCF, Ei-Sis interaction is lost and enhancement of VJ rearrangement is detected. Therefore, we propose that the interaction between Sis and Ei mediated by CTCF inhibits the VJ rearrangement and participates in the monoallelic silencing aspect of allelic exclusion regulation.