In studies on the cardiovascular disease risk among shift workers, smoking is considered to be a confounding factor. In a study of 239 shift and 157 daytime workers, it was found that shift work was prospectively related to increased cigarette consumption, indicating that smoking might be in the causative pathway; however, the number of study subjects was too low to warrant sound conclusions. Therefore, data from the Maastricht Cohort study were used to investigate the longitudinal relation between smoking and shift work in a much larger population. In this study, a total of 12,140 employees were followed for two years by means of self-administered questionnaires. The authors compared workers who normally worked during daytime hours only (74%) with those who worked other than day shifts (26%). Logistic regression analyses were performed, adjusting for demographic factors of age, gender, and educational level to evaluate the risk of starting to smoke (n = 25) in the group of non-smoking workers and the risk of quitting (n = 318) in the group of smoking workers. Logistic regression analysis showed a significant association between shift work and taking up smoking during the two-year follow-up (odds ratio: 1.46, p = 0.03). The risk to stop smoking was somewhat lower in shift workers (odds ratio: 0.91) but not statistically significant (p = 0.5). To conclude, this study showed that, independent of educational level, shift workers are more prone to start smoking. This finding might have important implications for studies on the health effects of shift workers and for possible interventions aimed at the reduction of the excess health risk among shift workers.