Smoking cessation reduces the risk of cardiovascular disease and improves clinical outcomes. We studied the effect of smoking cessation on plasma levels of α-klotho, which is an antiaging protein. We treated 28 smokers (male:female = 23:5, 46 ± 12 years) with varenicline (n = 14) or a transdermal nicotine patch (n = 14) as part of a 12-week smoking cessation program (the VN-SEESAW Study). Pulse rate, blood pressure, plasma levels of α-klotho, fibroblast growth factor (FGF)-19, FGF-21, hemoglobin (Hb), and expiratory carbon monoxide (CO) concentration were measured before and after the antismoking intervention. Smoking cessation significantly decreased pulse rate, α-klotho, Hb, and CO concentration, but not FGF-19 or FGF-21 in all subjects. On the contrary, body mass index significantly increased after the intervention. Changes in α-klotho levels (values at week 12 - values at week 0) were negatively associated with α-klotho levels at week 0 and positively associated with changes in Hb levels. In addition, the successful smoking cessation group (n = 21) showed significant reductions in pulse rate, systolic blood pressure, α-klotho, Hb, and CO concentration. In conclusion, smoking cessation significantly decreased serum levels of the antiaging molecule α-klotho. Our results are consistent with a previous report that an increase in α-klotho might be a compensatory response to smoking stress.