Migraine without aura (MWoA) is a major neurological disorder with unsatisfactory adherence to current medications. Acupuncture has emerged as a promising method for treating MWoA. However, the brain mechanism underlying acupuncture is yet unclear. The present study aimed to examine the effects of acupuncture in regulating brain connectivity of the key regions in pain modulation. In this study, MWoA patients were recruited and randomly assigned to 4 weeks of real or sham acupuncture. Resting-state functional magnetic resonance imaging (fMRI) data were collected before and after the treatment. A modern neuroimaging literature meta-analysis of 515 fMRI studies was conducted to identify pain modulation-related key regions as regions of interest (ROIs). Seed-to-voxel resting state-functional connectivity (rsFC) method and repeated-measures two-way analysis of variance were conducted to determine the interaction effects between the two groups and time (baseline and post-treatment). The changes in rsFC were evaluated between baseline and post-treatment in real and sham acupuncture groups, respectively. Clinical data at baseline and post-treatment were also recorded in order to determine between-group differences in clinical outcomes as well as correlations between rsFC changes and clinical effects. 40 subjects were involved in the final analysis. The current study demonstrated significant improvement in real acupuncture vs sham acupuncture on headache severity (monthly migraine days), headache impact (6-item Headache Impact Test), and health-related quality of life (Migraine-Specific Quality of Life Questionnaire). Five pain modulation-related key regions, including the right amygdala (AMYG), left insula (INS), left medial orbital superior frontal gyrus (PFCventmed), left middle occipital gyrus (MOG), and right middle cingulate cortex (MCC), were selected based on the meta-analysis on brain imaging studies. This study found that 1) after acupuncture treatment, migraine patients of the real acupuncture group showed significantly enhanced connectivity in the right AMYG/MCC-left MTG and the right MCC-right superior temporal gyrus (STG) compared to that of the sham acupuncture group; 2) negative correlations were established between clinical effects and increased rsFC in the right AMYG/MCC-left MTG; 3) baseline right AMYG-left MTG rsFC predicts monthly migraine days reduction after treatment. The current results suggested that acupuncture may concurrently regulate the rsFC of two pain modulation regions in the AMYG and MCC. MTG and STG may be the key nodes linked to multisensory processing of pain modulation in migraine with acupuncture treatment. These findings highlighted the potential of acupuncture for migraine management and the mechanisms underlying the modulation effects.