Introduction: This study aimed to assess the effect of controlled mouth breathing during the resting state using functional magnetic resonance imaging (fMRI). Methods: Eleven subjects participated in this experiment in which the controlled “, Nose”,and “, Mouth”,breathings of 6 s respiratory cycle were performed with a visual cue at 3T MRI. Voxel-wise seed-to-voxel maps and whole-brain region of interest (ROI)-to-ROI connectome maps were analyzed in both “, Nose>Mouth”,and “, Mouth>Nose”,contrasts. Results: As a result, there were more connection pairs in the “, Mouth”,breathing condition, i. e., 14 seeds and 14 connecting pairs in the “, Mouth>Nose”,contrast, compared to 7 seeds and 4 connecting pairs in the “, Nose>Mouth”,contrast (false discovery rate [FDR] of P<0. 05). Conclusion: The present study demonstrated that mouth breathing with controlled respiratory cycles could significantly induce alterations in functional connectivity in the resting-state network, suggesting that it can differently affect resting brain function,in particular, the brain can hardly rest during mouth breathing, as opposed to conventional nasal breathing.