Background: Cigarette smoking has a number of effects on the thyroid gland including the development of Graves' disease and thyroid multinodularity. However, the effect of smoking on thyroid function is more uncertain. Patients and methods: The present cross-sectional study included 6085 subjects (1744 smokers) without thyroxine medication and 441 subjects (92 smokers) with thyroxine medication that attended the 5th Tromsø study in 2001, and 460 subjects (114 smokers) that attended follow-up studies after the 5th Tromsø study. Serum TSH was measured in the 5th Tromsø study, and serum TSH, free T4, and free T3 in the follow-up studies. Comparisons between smokers and non-smokers were performed with a general linear model with age, BMI, (and gender) as covariables. Results: Serum TSH levels were significantly lower in the smokers than in the non-smokers, both in males (1.63 ± 0.88 vs. 1.95 ± 1.04 mIU/L [p < 0.01]), and in females (1.55 ± 0.86 vs. 1.86 ± 1.01 mIU/L [p < 0.01]). Serum free T4 and free T3 levels were significantly higher in smokers than non-smokers (14.0 ± 2.2 vs. 13.4 ± 2.4 pmol/L for free T4 [p < 0.05], and 3.89 ± 0.79 vs. 3.72 ± 0.67 pmol/L for free T3 [p < 0.01], males and females analyzed together). There was no association between number of cigarettes smoked and the serum TSH level. Conclusion: Smokers have lower serum TSH and higher free T4 and free T3 levels than non-smokers, which may be of importance when evaluating subjects with subclinical hypothyroidism.