Tyrosine hydroxylase (Th) encodes the rate-limiting enzyme in catecholamine biosynthesis, and the regulation of its transcription is critical for the specification and maintenance of catecholaminergic neuron phenotypes. For many genes, regulatory genomic DNA sequences that are upstream of the proximal promoter control expression levels as well as region-specific expression patterns. The regulatory architecture of the genomic DNA upstream of the Th proximal promoter, however, is poorly understood. In this study, we examined the 11 kb upstream nucleotide sequence of Th from nine mammalian species and identified five highly conserved regions. Using cultured human cells and mouse olfactory bulb tissue, chromatin immunoprecipitation (ChIP) assays show that these conserved regions recruit transcription factors that are established regulators of Th transcription (such as NURR1, PITX3, FOXA2, MEIS2, and PAX6). This analysis also identified a conserved binding site for CTCF, and functional studies in cultured human cells and ChIP assays with mouse tissue show that CTCF is a novel regulator of Th transcription in the forebrain. Together, the findings in this study provide key insights into the upstream regulatory genomic architecture and regulatory mechanisms controlling mammalian Th gene transcription.