Delta-like 1 (Dll1), which encodes the canonical Notch ligand DELTA1, plays a central role in neurogenesis during embryonic development and contributes to the organization of the adult nervous system. Dissecting the regulatory mechanisms that control Dll1 transcription, as well as their evolutionary modifications, is essential to understand the genetic basis of the brain diversity observed among species, particularly within our evolutionary lineage. In this work, we focused on conserved non-coding elements with potential enhancer activity. We tested their regulatory capacity through reporter assays in transgenic mice and mapped their spatial and temporal activity. To further assess their contribution in vivo, we generated enhancer knock-out models, where the loss of specific elements resulted in reduced Dll1 expression as measured by qPCR. RNA-seq showed that many genes potentially regulated by this pathway were either up- or downregulated. In addition, we modeled evolutionary changes by generating a knock-in mouse line in which the endogenous enhancer was replaced with the human orthologous sequence, carrying primate-specific substitutions. This experiment also pointed to transcriptional differences, suggesting that anthropoid-specific regulatory variants may influence Dll1 expression.