Epidemiological and clinical studies provide compelling support for a causal relationship between Helicobacter pylori infection and endothelial dysfunction, leading to vascular diseases. Clear biochemical evidence for this association is limited however. In the present study, we have conducted a comprehensive investigation of endothelial injury in bovine aortic endothelial cells (BAECs) induced by H.pylori-conditioned medium (HPCM) prepared from H.pylori 60190 (vacuolating cytotoxin A, Vac⁺). BAECs were treated with either unconditioned media, HPCM (0-25% v/v), or E.coli-conditioned media for 24 h, and cell functions monitored. Vac⁺ HPCM significantly decreased BAEC proliferation, tube formation, and migration (by up to 44%, 65%, and 28%, respectively). Post-treatment, we also observed sporadic zonnula occludens-1 (ZO-1) immunolocalization along the cell-cell border, and increased BAEC permeability to FD40 Dextran, indicating barrier reduction. These effects were blocked by NPPB (VacA inhibitor), and were not observed with conditioned media prepared from either VacA-deleted H.pylori or E.coli. The cellular mechanism mediating these events was also considered. Vac⁺ HPCM (but not Vac^-) reduced nitric oxide (NO) by over 50%, whilst SNAP, an NO donor, recovered all Vac⁺ HPCM-dependent effects on cell functions. We further demonstrated that laminar shear stress, an eNOS/NO stimulus in vivo, could also recover the Vac⁺ HPCM-induced decreases in BAEC functions. Conclusions: This study shows, for the first time, a significant pro-atherogenic effect of H.pylori secreted factors on a range of vascular endothelial dysfunction markers. Specifically, VacA-dependent reduction in endothelial NO is indicated in these events. The atheroprotective impact of laminar shear stress in this context is also evident.