Objective To explore the effect of acupuncture at Yanglingquan (GB 34) on white matter structure of stroke patients. Methods 6 stroke patients with left hemiplegia in recovery stage were recruited. The sham acupoint was as control, the diffusion tensor imaging (DTI) data in a 1.5 T SIEMENS scanner before and after acupuncture at Yanglingquan point was collected. Differences between the two conditions were compared with the Tract-Based Spatial Statistics (TBSS), which is one of FSL tools. Results The brain areas included left precentral gyrus, postcentral gyrus, middle temporal gyrus, inferior temporal gyrus, supplementary motor area, inferior frontal gyrus, internal capsule, fornix and bilateral inferior parietal gyrus, in which the verum acupuncture had stronger effect on fractional anisotropy (FA) than the sham acupuncture. White matter tract such as pyramidal tract, arcuate fasciculus, corpus callosum and other inter-cortex association fibers passed these brain areas. The fiber tracts and brain areas were found to be involved with multiple brain networks of motor compensation, somatosensory, language and cognition. Conclusion Compared with the sham point, acupuncture at Yanglingquan point can change the microstructure of stroke patients' white matter, mainly in the inaffected side and related to multiple functional networks.