Although a substantial body of previous functional magnetic resonance imaging (fMRI) studies have revealed different brain responses to external stimuli in chronic cigarette smokers compared with nonsmokers, only a few studies assessed brain spontaneous activity in the resting state in chronic smokers. The aim of this study was to investigate alterations of brain activity during the resting state in chronic smokers using fractional amplitude of low-frequency fluctuation (fALFF). In the present study, 55 smokers and 49 healthy nonsmokers were included. All the subjects underwent resting-state fMRI scans and the data were analyzed by the fALFF approach. The smokers showed significantly decreased fALFF in the left precuneus, right inferior temporal and occipital gyrus(ITG/IOG), while significantly increased fALFF in the right caudate. Subsequent correlation analysis revealed that the fALFF values of the left precuneus and right ITG/IOG were positively correlated with years of smoking across the smokers. This resting-state fMRI study suggests that the changed spontaneous neuronal activity, as reflected by the fALFF, in these regions may be implicated in the underlying the pathophysiology of smoking.