Eukaryotic chromosomes are not randomly distributed in the interphase nucleus, but instead occupy distinct territories. Nonetheless, the genome-wide relationships of gene regulation to gene nuclear location remain poorly understood in yeast. In the three-dimensional view of gene regulation, we found that a considerable number of transcription factors (TFs) regulate genes that are colocalized in the nucleus. Colocalized TF target genes are more strongly coregulated compared with the other TF target genes. Target genes of chromatin regulators are also colocalized. These results demonstrate that colocalization of coregulated genes is a common process, and three-dimensional gene positioning is an important part of gene regulation. Our findings will have implications in understanding nuclear architecture and function.