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Background Electroacupuncture (EA) is currently one of the most popular acupuncture modalities. However, the continuous stimulation characteristic of EA treatment presents challenges to the use of conventional functional Magnetic Resonance Imaging (fMRI) approaches for the investigation of neural mechanisms mediating treatment response because of the requirement for brief and intermittent stimuli in event related or block designed task paradigms. A relatively new analysis method, functional connectivity fMRI (fcMRI), has great potential for studying continuous treatment modalities such as EA. In a previous study, we found that, compared with sham acupuncture, EA can significantly reduce Periaqueductal Gray (PAG) activity when subsequently evoked by experimental pain. Given the PAG's important role in mediating acupuncture analgesia, in this study we investigated functional connectivity with the area of the PAG we previously identified and how that connectivity was affected by genuine and sham EA. Results Forty-eight subjects, who were randomly assigned to receive either genuine or sham EA paired with either a high or low expectancy manipulation, completed the study. Direct comparison of each treatment mode's functional connectivity revealed: significantly greater connectivity between the PAG, left posterior cingulate cortex (PCC), and precuneus for the contrast of genuine minus sham; significantly greater connectivity between the PAG and right anterior insula for the contrast of sham minus genuine; no significant differences in connectivity between different contrasts of the two expectancy levels. Conclusions Our findings indicate the intrinsic functional connectivity changes among key brain regions in the pain matrix and default mode network during genuine EA compared with sham EA. We speculate that continuous genuine EA stimulation can modify the coupling of spontaneous activity in brain regions that play a role in modulating pain perception., National Center for Complementary and Alternative Medicine (U.S.) (PO1-AT002048), National Center for Complementary and Alternative Medicine (U.S.) (R01AT005280), National Center for Complementary and Alternative Medicine (U.S.) (R21AT00949), National Center for Complementary and Alternative Medicine (U.S.) (KO1AT003883), National Center for Complementary and Alternative Medicine (U.S.) (R21AT004497), National Center for Complementary and Alternative Medicine (U.S.) (K24AT004095), National Center for Research Resources (U.S.) (Clinical Research Center Biomedical Imaging Core, M01-RR-01066), National Center for Research Resources (U.S.) (Clinical Research Center Biomedical Imaging Core, UL1 RR025758-01), National Center for Research Resources (U.S.) (Center for Functional Neuroimaging Technologies, P41RR14075)