OBJECTIVE: Cigarette smoking is an important risk factor for many diseases. This study aimed to evaluate whether cigarette smoking is associated with changes in the thiol/disulfide homeostasis (TDH), a novel biomarker of systemic oxidative stress. PATIENTS AND METHODS: Eighty-four smokers and 86 non-smoking healthy volunteers were enrolled. Serum native thiol, disulfide and total thiol levels, disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios were analyzed using a new colorimetric method. Carbon monoxide (CO) levels were measured by a piCO smokerlyzer instrument. RESULTS: The native, total, and native/total thiol levels of smoking patients were significantly lower (p<0.001 for each), and disulfide, disulfide/native thiol, and disulfide/total thiol levels were significantly higher in smokers than the healthy controls (p<0.001 for each). The CO levels of all study participants were negatively correlated with native thiol (r= -0.627, p<0.001), total thiol (r= -0.569, p<0.001), native thiol/total thiol (r= -0.515, p<0.001), and positively correlated with disulfide (r=0.398, p<0.001), disulfide/native thiol (r=0.515, p<0.001) and disulfide/total thiol (r=0.515, p<0.001) levels. CONCLUSIONS: To our knowledge, this investigation is the first in the literature that investigated TDH in cigarette smokers. Our results show that cigarette smoking may lead to oxidative stress and TDH shifts through disulfide side compared to the healthy group. Further studies with larger sample size are needed to confirm our results for showing the changes in TDH to contribute to the clinical practice.