GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate limiting step for the de novo production of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS). Interestingly, the GTP-CH1-BH4 pathway is emerging as a potential regulator in a number of pathologies associated with nitric oxide (NO) over production. GTP-CH1 activity can be inhibited by BH4 through its protein-protein interactions with GTP-CH1 regulatory protein (GFRP) and transcriptional and post translational modification of both GTP-CH1 and GFRP have been reported in response to proinflammatory stimuli but the functional significance of GFRP/GTP-CH1 interactions on NO pathways has not yet been demonstrated. We aimed to investigate whether over expression of GFRP could affect NO production in living cells. We have over-expressed N-terminally -Myc tagged recombinant human GFRP in the murine endothelial cell line, sEnd 1. BH4 and nitrite levels were measured both basally and following stimulation with proinflammatory cytokines and lipopolysaccharide. GFRP over expression had no significant effect on basal BH4 nor NO levels but significantly attenuated the rise in BH4 and NO observed following cytokine stimulation of cells. This study demonstrates that GFRP can play a direct regulatory role in iNOS mediated NO synthesis and suggests that the allosteric regulation of GTP-CH1 activity by GFRP may be an important mechanism regulating BH4 and NO levels in vivo.