Background Previous neuroimaging studies primarily focused on the spatial distribution of acupuncture needling stimulation. However, a salient feature of acupuncture was its long-lasting effect. This study attempted to detect the spatial-temporal neural responses evoked by acupuncture at an analgesia acupoint ST36 by using magnetoencephalography. To further verify its functional specificity, we also adopted acupuncture at Pericardium 6 and nonacupoint as separated controls. Methods Forty-two college students, all right-handed and acupuncture naive, participated in this study. Every participant received only one acupoint stimulation, resulting in 14 subjects in one group. Both magnetoencephalography data (151-channel whole-head system) and structural functional magnetic resonance imaging data (3D sequence with a voxel size of 1 nnm(3) for anatomical localization) were collected for each subject. All processing procedures were performed in BrainStorm Toolbox. Results Acupuncture at ST36 showed a significantly time-varied brain activities with different onset time. Our results presented that acupuncture at different acupoints (or comparing with nonacupoint) can specifically induce neural responses in different brain areas acupuncture at ST36 can specifically induce the neural responses of pain-inhibition areas, while acupuncture at PC6 can specifically induce the activities of the insula and amygdala. Conclusions In the present study, we attempted to detect the temporal neural responses underlying the functional specificity of acupuncture at ST36, using acupoint belonging to different meridians and non-acupoint with efficacy-irreverent as separate controls. The specific neural substrates involving acupuncture at different acupoints may be related to its functional specificity in clinical settings. Chin Med J 2011;124(8):1229-1234