Significance The apparent importance of promoter:enhancer looping is well established; however, the molecular mechanisms of these interactions in gene activation vs. gene repression remain to be fully elucidated. Here, we report that LIM domain-binding protein 1 (LDB1) can function in transcriptional enhancer-mediated gene activation mainly at the level of transcription initiation by regulating promoter:enhancer looping, consequent to the recruitment to basic helix-loop-helix–bound enhancers in pituitary corticotrope cells. Intriguingly, LDB1 also mediates promoter:enhancer looping required for target gene repression, acting at the level of promoter pausing, by recruiting metastasis-associated 1 family, member 2 to these repressive enhancers. These findings shed light on a regulatory aspect of the molecular function of LDB1, providing a putative mechanism of enhancer-dependent transcriptional repression.