Over the past decades the search for disease causing variants has been focusing exclusively on the coding genome. This highly selective approach has been extremely successful resulting in the identification of thousands of disease genes, but ignores the functional and therefore disease relevance of the rest of the genome. Dropping sequencing costs and new high-throughput technologies such as ChIP-seq and chromosome conformation capture have opened new possibilities for the systematic investigation of the non-coding genome. These data have revealed the importance of non-coding DNA in fundamental processes such as gene regulation and 3D chromatin folding. Research into the principles of chromatin folding has revealed a domain structure of the genome, called topologically associated domains that provide a scaffold for enhancer promoter contacts. Non-coding mutations that affect regulatory elements can affect gene regulation by a loss of function, resulting in reduced gene expression, or a gain of function resulting in gene mis- or overexpression. Structural variations such as deletions, inversions or duplications have the potential to disturb normal chromatin folding. This may lead to the repositioning or disruption of topological associating domains and the relocation of enhancer elements with consecutive gene misexpression. Several recent studies highlight this as important disease mechanisms in developmental disorders and cancer. Therefore, the regulatory landscape of the genome has to be taken into consideration when investigating the pathology of human disease. In this review, we will discuss the recent discoveries in the field of non-coding variation, gene regulation, 3D genome architecture, and their implications for human genetics.