Background: The periaqueductal gray (PAG) dysfunction was recognized in migraine, and the altered dysfunction of PAG subregions were not totally detected up to now. The aim of this study is to investigate the altered functional connectivity of PAG subregions in EM patients. Methods: The brain structural images and resting state functional MR imaging (rs-fMRI) data were obtained from 18 normal controls (NC) and 18 EM patients on 3.0 T MR system. Seven subregions of PAG were classified as bilateral ventrolateral PAG (vlPAG), lateral PAG (lPAG), dorsolateral PAG (dlPAG) and dorsomedial PAG (dmPAG). The functional connectivity maps of each PAG subregion were calculated, and Two sample t-test was applied with age and sex as covariables. Results: Bilateral vlPAG and left dlPAG presented decreased functional connectivity, and the other subregions (bilateral lPAGs, right dlPAG and dmPAG) showed no significant altered functional connectivity in EM compared with NC. The brain regions with decreased functional connectivity mainly located in bilateral prefrontal cortex(PFC), middle temporal gyrus, primary motor area (PMA) and supplementary motor area (SMA) and right ventrolateral PFC (vlPFC) in EM patients in this study. Disease duration was positively related to the functional connectivity of bilateral vlPAG on the bilateral thalamus and putamen, left pallidum and right medial orbitofrontal gyrus in EM patients. Conclusion: The present study suggested that the dysfunction of bilateral vlPAG and left dlPAG presented in EM, and functional evaluation of PAG subregions may be help for the diagnosis and understanding of EM pathogenesis.