Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution. ; This study was supported by the Spanish and Andalusian Governments (BFU2013-41322-P and Proyecto de Excelencia BIO-396 to J.L.G.-S., BFU2013-43213-P to P.B., BFU2014-55738-REDT to J.L.G.-S. and P.B., and BFU2011-22928 to J.C. and M.L.-M.). EU-FP7-PEOPLE-2011-CIG Grant 303904 “EPAXIALMYF5KO” also supported J.C. ; Peer Reviewed