Objectives Cigarette smoking had been recorded as the main cause of impaired endothelium-dependent vasodilation in smokers by reducing nitric oxide (NO), a production of endothelial nitric oxide synthase (eNOS). However, the mechanism of NO impairment via eNOS activity is unclear until now. In this study, cell passage is suggested to be a relevant factor to eNOS expression under cigarette smoking stress. Methods Bovine aortic endothelial cells (BAECs) were chosen as the research subject with passages ranking from 6 to 9 (6P to 9P). After exposure of cigarette smoking extract (CSE) solution, MTT assay and Western blot method were performed to check the cell viability as well as eNOS protein concentration. In these experiments, four concentrations of CSE at 0.5, 1, 2, and 4% were selected for treatment. Results Our results showed that cells almost died at 4% of CSE. Besides, eNOS protein mass had a linear decrease under the increase of CSE concentration. In addition, the effect of CSE on eNOS expression was dissimilar between different passages. Conclusions This study indicated that CSE had effect on both cell viability and eNOS expression. Besides, a reduction in protein mass was matched with the decrease of cell viability due to CSE tress. Last but not least, the response of eNOS protein to different concentration of CSE at different passages was disparate, making the hypothesis about cell passage related inhibition of eNOS caused by CSE solution.