Objectives Netrin-1 is a laminin like protein that has recently been suggested to involve in the pathophysiology of atherosclerosis and treatment targets. The relationship between netrin-1 and atherosclerosis has not been evaluated in a human research previously and the effect of smoking on plasma netrin-1 levels as well. In this study, we aimed to evaluate plasma netrin-1 levels in smokers and non-smokers and the impact of smoking on netrin-1 levels. Our second aim was to evaluate the existence of peripheral artery disease in these groups by means of ankle brachial index (ABI) measurements and the relation between plasma netrin-1 levels and peripheral artery disease. Design and Methods Smoker and non-smoker male patients were contributed in the study, 112 males in each group. ABI of all patients was determined with an automatic device and plasma netrin-1 levels were quantified with an enzyme-linked immunosorbent assay. Netrin-1 levels were compared between smoker and non-smoker cases, and the cases with and without peripheral arterial disease. Results Plasma netrin-1 levels of smokers (4.54 ± 2.87 pg/mL) were significantly higher than in non-smokers (3.81 ± 1.26 pg/mL) (p = 0.015). Similarly, netrin-1 levels of cases with peripheral arterial disease (6.21 ± 2.16 pg/mL) were significantly higher than the ones without peripheral arterial disease (4.07 ± 2.16 pg/mL) (p = 0.002). Amount of smoking (packet ⋅ year) and ABI were found to be significantly related with netrin-1 levels. The relationship between PAD and netrin-1 levels remained significant after adjusting for smoking status. Conclusion We can conclude that plasma netrin-1 levels significantly increase in smokers and peripheral artery disease is significantly related with netrin-1 levels after adjusting for smoking status.