Correct temporal and spatial patterns of gene expression are required to establish unique cell types. Several levels of genome organization are involved in achieving this intricate regulatory feat. Insulators are elements that modulate interactions between other cis-acting sequences and separate chromatin domains with distinct condensation states. Thus, they are proposed to play an important role in the partitioning of the genome into discrete realms of expression. This review focuses on the roles that insulators have in vivo and reviews models of insulator mechanisms in the light of current understanding of gene regulation.