Lower troponin concentrations measured in smokers in a healthy population raise the question of whether a lower troponin threshold should be considered for tobacco users. We aim to evaluate differences in troponin levels according to the smoking status in healthy young adults. Participants aged 25–41 years were enrolled in a population-based observational study. The smoking status was self-assessed, and participants were classified as never-, past-, and current smokers. Pack-years of smoking were calculated. High-sensitivity cardiac troponin I (hs-cTnI) concentrations were measured from thawed blood samples, and associations were assessed using multivariable linear regression analyses. We included 2155 subjects in this analysis. The mean (SD) age was 35.4 ± 5.22 years; 53% were women. The median hs-cTnI levels across smoking status categories were 0.70 (interquartile range 0.43–1.23) ng/L in never smokers (n = 1174), 0.69 (interquartile range 0.43–1.28) ng/L in past smokers (n = 503), and 0.67 (interquartile range 0.41–1.04) ng/L in current smokers (n = 478), p = 0.04. The troponin levels remained significantly lower in current smokers after adjustment for potential confounders (β-coefficient [95%CI] of −0.08 [−0.25; −0.08], p < 0.001). Our results confirm that current smokers have lower hs-cTnI levels than past or never smokers, with a significant dose–response relationship among current smokers. The absolute differences in hs-cTnI levels were small.