Background Noninvasive functional magnetic resonance imaging (fMRI) techniques have opened a "window" into the brain, allowing us to investigate the anatomical and physiological function involving acupuncture needling. Imaging its sustained effect rather than acute effect on the brain networks may further help elucidate the mechanisms by which acupuncture achieves its therapeutic effects. In this study, we aimed to investigate the functional brain networks during the post-resting state following acupuncture at KI3 in comparison with acupuncture at GB40. Methods Needling at acupoints GB40 and KI3 was performed in twelve subjects. Six minutes of scanning at rest were adopted before and after acupuncture at different acupoints. Then we divided the whole brain into 39 regions and constructed functional brain networks during the post-acupuncture resting states (PARS). Results For direct comparisons, increased correlations during post-resting state following acupuncture at KI3 compared to resting state (RS) were primarily located between the dorsolateral prefrontal cortex (DLPFC) and post temporal cortex, ventromedial prefrontal cortex (vmPFC) and post temporal cortex. These brain regions were all cognitive-related functions. In contrast, the increased connections between the anterior insula and temporal cortex mainly emerged following acupuncture at GB40 compared with the RS. Conclusions The present study demonstrates that acupuncture at different acupoints belonging to the same anatomic segment can exert different modulatory effects on the reorganizations of post-acupuncture RS networks. The heterogeneous modulation patterns between two conditions may relate to the functional specific modulatory effects of acupuncture. Chin Med J 2012;125(15):2694-2700