Background Lung cancer screening may provide a new opportunity for attempts to quit among smokers or might delay smoking cessation, but studies to date failed to provide evidence for this. This study investigated the effect of lung cancer screening on smoking abstinence in male smokers participating in the Dutch–Belgian randomised controlled lung cancer screening trial (NELSON trial). Methods In the NELSON trial, 50- to 75-year-old participants at high risk for developing lung cancer were randomised to either lung cancer screening or no screening. Smoking behaviour was evaluated in two random samples of male smokers in the screen (n=641) and control arm (n=643) before (T0) and 2&emsp14;years after randomisation (T1). In addition, the data were also analysed by intention-to-treat (ITT) analysis, as recommended in smoking cessation intervention trials, although non-response in screening trials can also be due to reasons other than continued smoking. Results Almost 17% (16.6%) of the trial participants quit smoking, which is higher than the 3–7% found in the general adult population. However, screening was associated with a lower prolonged abstinence rate (14.5%) compared with no screening (19.1%) (OR 1.40, 95% CI 1.01 to 1.92; p<0.05). No stastistically significant difference was found after performing an ITT analysis. Conclusions This study showed that all trial participants were inclined to stop smoking more than average, which suggests that screening is a teachable moment to improve smoking behaviour. In those who underwent screening the smoking abstinence rate was significantly lower than for the control group, although the difference was modest. After ITT analysis this difference was no longer observed. Clinical trial number ISRCTN63545820.