Aims: Tobacco smoking is known to increase the long-term risk of developing Type 2 diabetes mellitus, but the mechanisms involved are poorly understood. This observational, cross-sectional study aims to compare measures of insulin sensitivity and β-cell function in current, ex- and never-smokers. Methods: The study population included 1246 people without diabetes (mean age 44 years, 55% women) from the EGIR-RISC population, a large European multicentre cohort. Insulin sensitivity was measured using a hyperinsulinaemic, euglycaemic clamp and the homeostatic model assessment – insulin resistance (HOMA-IR) index. Two β-cell function parameters were derived from measures during an oral glucose tolerance test: the early insulin response index and β-cell glucose sensitivity. Additionally, the areas under the curve during the oral glucose tolerance test were calculated for glucose, insulin and C-peptide. Results: According to smoking habits, there were differences in insulin sensitivity, which was lower in women who smoked, and in β-cell glucose sensitivity, which was lower in men who smoked, but these associations lost significance after adjustment. However, after adjustment, the areas under the glucose and the C-peptide curves during the oral glucose tolerance test were significantly higher in men who smoked. Conclusions: Smoking habits were not independently associated with insulin sensitivity or β-cell function in a healthy middle-aged European population. Health-selection bias, methodological shortcomings or a true lack of causal links between smoking and impaired insulin sensitivity/secretion are possible explanations. The mechanisms behind the observed increased glucose and C-peptide areas under the curve during the oral glucose tolerance test in male smokers need to be further evaluated.