Nicotine has been known to play a pathogenic role in various cardiovascular disorders. However, the definite mechanism of nicotine-mediated endothelial dysfunction in vivo remains unclear because hemodynamic factor in most of in vitro studies was excluded. In this study, we investigated how nicotine affects human umbilical vein endothelial cells (HUVECs), from views of inflammatory and hemostatic responses of the cells, under a hemodynamic environment as occurred in vivo. Our results showed that both inflammation, reflected by production of reactive oxygen species and efficacy of monocytes adhesion, and hemostatic expression of HUVECs were abnormally enhanced after treated with 10−4 M nicotine and 12 dynes cm−2 laminar shear stress (LSS) simultaneously for 24 h, and that the protein expression levels of VCAM-1, ICAM-1, and PAI-1 were significantly enhanced 1.3-, 2- and 2-fold (p < 0.05 for each), respectively, as compared to the group with nicotine alone; 2.2-, 3- and 4.2-fold (p < 0.05 for each), respectively, as compared to the group with LSS alone. We reasoned that those irregular expressions were resulted from the reduction of endothelial nitric oxide synthase that was initially caused by nicotine exposure and exacerbated due to LSS treatment. Furthermore, all the impaired responses can be alleviated by use of 1 μg mL−1 recombinant tissue plasminogen activator, implicating that the irregular inflammation may be due to thrombosis.