ObjectiveMotor recovery is crucial in stroke rehabilitation, and acupuncture can influence recovery. Neuroimaging and machine learning approaches provide new research directions to explore the brain functional reorganization and acupuncture mechanisms after stroke. We applied machine learning to predict the classification of the minimal clinically important differences (MCID) for motor improvement and identify the neuroimaging features, in order to explore brain functional reorganization and acupuncture mechanisms for motor recovery after stroke.MethodsIn this study, 49 patients with unilateral motor pathway injury (basal ganglia and/or corona radiata) after ischemic stroke were included and evaluated the motor function by Fugl–Meyer Assessment scores (FMA) at baseline and at 2-week follow-up sessions. Patients were divided by the difference between the twice FMA scores into one group showing minimal clinically important difference (MCID group,n= 28) and the other group with no minimal clinically important difference (N-MCID,n= 21). Machine learning was performed by PRoNTo software to predict the classification of the patients and identify the feature brain regions of interest (ROIs). In addition, a matched group of healthy controls (HC,n= 26) was enrolled. Patients and HC underwent magnetic resonance imaging examination in the resting state and in the acupuncture state (acupuncture at the Yanglingquan point on one side) to compare the differences in brain functional connectivity (FC) and acupuncture effects.ResultsThrough machine learning, we obtained a balance accuracy rate of 75.51% and eight feature ROIs. Compared to HC, we found that the stroke patients with lower FC between these feature ROIs with other brain regions, while patients in the MCID group exhibited a wider range of lower FC. When acupuncture was applied to Yanglingquan (GB 34), the abnormal FC of patients was decreased, with different targets of effects in different groups.ConclusionFeature ROIs identified by machine learning can predict the classification of stroke patients with different motor improvements, and the FC between these ROIs with other brain regions is decreased. Acupuncture can modulate the bilateral cerebral hemispheres to restore abnormal FCviadifferent targets, thereby promoting motor recovery after stroke.Clinical trial registrationhttps://www.chictr.org.cn/showproj.html?proj=37359, ChiCTR1900022220.