Accumulating evidence suggested that overweight and smoking often co-exist. However, current neuroimaging researches have almost always studied smoking or overweight status separately. Here we sought to investigate the neurobiological mechanisms of this comorbid association, by detecting spontaneous brain activity changes associated with smoking and weight status separately and collectively. We used 2 × 2 factorial design and included the following four groups: overweight/normal-weight smokers ( = 34/ = 30) and overweight/normal-weight non-smokers ( = 22/ = 24). The spontaneous brain activity among the four groups was comparable using an amplitude of low-frequency fluctuation (ALFF) method based on resting-state fMRI (rs-fMRI). Furthermore, correlation analyses between brain activity changes, smoking severity and BMI values were performed. A main effect of smoking was discovered in the default mode network (DMN) and visual network related brain regions. Moreover, overweight people had high ALFF value in the brain regions associated with reward and executive control. More importantly, smoking and overweight both affected brain activity of the middle temporal gyrus (MTG), but the effect was opposite. And the brain activity of MTG was negatively correlated with smoking years, pack year and BMI value. These results suggest that smoking and overweight not only affect spontaneous brain activity alone, but also paradoxically affect spontaneous brain activity in the MTG. This suggests that we need to control for weight as a variable when studying spontaneous brain activity in smokers. Besides, this interaction may provide a neurological explanation for the comorbidity of overweight and smoking and a target for the treatment of comorbid populations.