BACKGROUND: The amygdala is a key region in emotion processing, and studies have suggested that amygdala-frontal functional connectivity deficits could be modulated by antidepressants in major depressive disorder (MDD). Transcutaneous vagus nerve stimulation (tVNS), a non-invasive, peripheral neuromodulation method at the ear, has shown promising results in treating major depressive disorder (MDD) in several pilot studies. However, the neural mechanism underlying tVNS treatment of depression has not been fully investigated. In this study, we investigated how tVNS can modulate the amygdala-lateral prefrontal network resting state functional connectivity (rsFC) in mild or moderate major depressive disorder (MDD) patients. METHODS: Forty-nine MDD patients were recruited and received tVNS or sham tVNS (stVNS) treatments for four weeks. Resting state fMRI scans were applied before and after treatments. RESULTS: After 1 month of tVNS treatment, the 24-item Hamilton Depression Rating Scale (HAMD) scores were reduced significantly in the tVNS group as compared with the sham tVNS group. The rsFC in the tVNS group between the right amygdala and left dorsolateral prefrontal cortex was increased compared with sham tVNS. All the rsFC increases were also associated with HAMD reduction as well as reductions in the anxiety and retardation HAMD subscales. CONCLUSIONS: tVNS can significantly modulate the amygdala-lateral prefrontal rsFC of MDD patients; our results provide insights into the brain mechanism of tVNS treatment for MDD patients.