Periodontal disease is a highly prevalent chronic inflammatory disease and is associated with complex microbial infection in the subgingival cavity. Recently, American Heart Association supported a century old association between periodontal disease and atherosclerotic vascular disease. We have recently shown that polybacterial periodontal infection led to aortic atherosclerosis and modulation of lipid profiles; however the underlying mechanism(s) has not been yet demonstrated. Altered nitric oxide (NO) synthesis and tetrahydrobiopterin (BH4), a cofactor for nitric oxide synthases (NOS) has long been shown to be associated with vascular dysfunction and gastrointestinal motility disorders. We sought to examine the mechanism of periodontal infection leading to altered vascular and gastrointestinal smooth muscle relaxation, focusing on the BH4/nNOS pathways. In addition, we also have investigated how the antioxidant system (NRF2-Phase II enzyme expression) in vascular and GI specimens is altered by oral infection. Eight week old male ApoEnull mice were either sham-infected or infected orally for 16 weeks with a mixture of major periodontal bacteria Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia to induce experimental periodontitis. Serum, vascular (mesenteric), stomach, and colon specimens were collected at the end of periodontal pathogen infection. Bacterial infection induced significant (p<0.05) reductions in the levels of BH4,in ratio of BH4:BH2+B and also in nitric oxide levels compared to sham-infected controls. In addition, we identified a significant (p<0.05) reduction in eNOS dimerization, nNOS dimerization and protein expression of BH4 biosynthesis enzymes; GCH-1, DHFR and NRF2 & Phase II enzymes in infected mice versus controls in both mesenteric artery and colon tissues. However, we found no differences in nNOS/BH4 protein expression in stomach tissues of infected and sham-infected mice. This suggests that a polybacterial infection can cause significant changes in the vascular and colonic BH4/nNOS/NRF2 pathways which might lead to impaired vascular relaxation and colonic motility.