Background Although about one third of smokers tried to quit smoking within the last 12 months, only 3-5% remain abstinent at 6 months. This indicates the presence of long-term modifications in brain networks related to smoking. Using functional magnetic resonance imaging (fMRI), we compared ex-smokers to active and non-smokers to explore these modifications. Methods 18 non-smokers (29.51 ± 6.70 years, 11 females) 14 smokers (≥10 cigarettes a day > 2 years, 29.31 ± 6.04 years, 10 females) and 14 ex-smokers (>1 year of quitting 30.5 ± 5.70 years, 10 females) were recruited through local and Internet advertising. A block-design fMRI study was performed contrasting smoking cue videos versus control videos. Data analyses include task-related general linear model (GLM), seed based functional connectivity, voxel-based morphometry (VBM) of grey matter and tract based spatial statistics (TBSS) of white matter. Results Smoking cue videos versus control videos activated notably the right anterior insula for the contrast ex-smokers versus smokers, an effect correlating with the accumulated dose of nicotine. In addition, ex-smokers, compared to controls and similarly to smokers, showed a persistent decrease in the functional connectivity between anterior insula and anterior cingulate cortex (ACC)...