GTP cyclohydrolase I (GCH‐1) is the rate limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4) that is an essential cofactor for Nitric Oxide Synthase and aromatic amino acid hydroxylase. To explore the cell biology of GCH‐1 and determine the proteins interacting with GCH‐1, we cloned rat GCH‐1 linked with FLAG and established a stable HEK293 cell line expressing GCH‐1. We confirmed GCH‐1 expression by western blot. FLAG‐GCH‐1 associated proteins were immunoprecipitated and analyzed by ESI‐nano‐LC/MS/MS. 38 proteins were found to interact with GCH‐1. Analysis revealed that many proteins associated with GCH‐1 were involved in cellular signaling, organization, protein expression/trafficking, metabolism and apoptosis. 21 of 38 Proteins were related to cellular signaling pathways including receptors (olfactory receptors), protein kinases (MRCKƒ8 4, LATS1) and transcription factors (SNF2L1, ATF‐6ƒ8 4). GCH‐1 was found to associate with dihydropyrimidine dehydrogenase, directly related to its function. Surprisingly p47‐phox was also observed to associate with GCH‐1. Although GCH‐1 is considered cytosolic, the proteins associated with GCH‐1 were also found in the membrane, nucleus and other subcellular compartments (mitochondria and Golgi). This study is the first to provide a comprehensive analysis of GCH‐1 interactome and offer new insight into potential mechanisms of diseases related to GCH‐1.