With advances in genetic science, the dynamic structure of eukaryotic genome is considered as basis of gene expression regulation. Long-distance communication between regulatory elements and target promoters is critical and the mechanisms responsible for this connection are just starting to emerge. Chromatin insulators are key determinants of proper gene regulation and precise organization of chromosomal structures. These elements are DNA-protein complexes which can mediate interactions within and between chromosomes. In doing so, they can bring close together regulatory sequences located at large distances from each other. In addition, insulators may be important in remodeling the architecture of chromosomes in the cell cycle. Here, we review recent data revealing the structure and properties of these DNA-protein complexes, their global role in regulation of transcription, and the mechanisms by which misregulation of their function leads to various human diseases such as cancers and autoimmune diseases.